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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
</ul>
<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong> Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions:</strong> Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
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<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p>Diagenode <strong>Tagmentation Buffer (2x)</strong> is the recommended reagent to perform any tagmentation reactions. It can be used in combination with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase)</a> on DNA or chromatin samples, as half of the total volume reaction like in ATAC-seq protocol.</p>
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'info1' => '<p><span style="text-decoration: underline;">ATAC-seq experiments: </span></p>
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<li>After cell lysis and nuclei isolation, the nuclei pellets can be incubated with the following mix for 1 reaction:</li>
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<td style="width: 326px;">Tagmentation Buffer (2x)</td>
<td style="width: 114px; padding-left: 30px;">25 µl</td>
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<td style="width: 114px; padding-left: 30px;">2.5 µl</td>
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<td style="width: 326px;"><span>Digitonin 1%</span></td>
<td style="width: 114px; padding-left: 30px;">0.5 µl</td>
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<td style="width: 114px; padding-left: 30px;">0.5 µl</td>
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<td style="width: 114px; padding-left: 30px;"> 5 µl</td>
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<p><em>* The number of nuclei per reaction will depend on the ATAC-seq experimental design. Successful tagmentation with the proposed protocol has been performed on 50,000 nuclei per reaction. </em></p>
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<li>The reaction is then incubated 30 minutes at 37°C.</li>
<li>The tagmentation reaction can then be stopped by addition of 250 µl of DNA Binding buffer from Diagenode MicroChIP DiaPure Columns (Cat. No. C03040001).</li>
<li>The tagmented libraries can then be purified using the MicroChIP DiaPure Columns (Cat. No. C03040001), and amplified.</li>
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<p>The <strong>24 UDI for tagmented libraries</strong> includes 24 primer pairs for unique dual-indexing allowing the multiplexing of up to <b>24 samples </b>for sequencing on Illumina platforms. These UDI are designed and validated to be used with <a href="https://www.diagenode.com/en/p/uchipmentation-for-histones-24-rxns">μChIPmentation for Histones</a> (Cat. No. C01011011), <a href="https://www.diagenode.com/en/p/chipmentation-kit-for-histones">ChIPmentation Kit for Histones</a> (Cat. No. C01011009), <a href="https://www.diagenode.com/en/p/tag-kit-for-chipmentation-24">TAG Kit for ChIPmentation</a> (Cat. No. C01011030), <a href="https://www.diagenode.com/en/categories/atac-seq">ATAC-seq Kit</a> (Cat. No. C01080002). The 24 UDI for tagmented libraries are compatible with other <b>tagmentation</b><b>-based library preparation </b>protocols, such as <a href="https://www.diagenode.com/en/categories/cutandtag">CUT&Tag</a> technologies.</p>
<p>3 sets of UDI for tagmented libraries are available:</p>
<p><strong>24 UDI for tagmented libraries - Set I</strong><br /> <a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set2">24 UDI for tagmented libraries - Set II</a><br /><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set3" target="_blank">24 UDI for tagmented libraries - Set III</a><br /><br /></p>
<p><span>Each set can be used for library multiplexing up to 24. All sets can be used simultaneously for library multiplexing up to 72.</span></p>
<p>Features:</p>
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<li>Allow for <b>identification of index hopping</b></li>
<li>Compatibility: <b>tagmentation</b><b>-based library preparation protocols</b></li>
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'info1' => '<p>The <b>24 UDI (Unique dual indexes) for </b><b>tagmented</b><b> libraries – Set I </b>is compatible with any <b>tagmentation</b><b>-based library preparation </b>protocols, such as <strong>ChIPmentation</strong>, <b>ATAC-seq</b> or <b>CUT&Tag</b> technologies.</p>
<p>The <b>24 UDI for </b><b>tagmented</b><b> libraries </b>provides combinations of barcodes where each barcode is uniquely attributed to one sample. This is a great tool to identify mistakes during index sequencing. A phenomenon, known as index hopping, can lead to misattribution of some reads to the wrong sample. This is particularly frequent with the NovaSeq6000, and thus the use of Unique Dual Indexing (UDI) is highly recommended when using this sequencer.</p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/UDI-for-tagmented-fig1.png" /></center>
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<p><small><strong>Figure 1. Sequencing profiles of µChIPmentation libraries generated with 24 UDI for Tagmented libraries</strong> Chromatin preparation and immunoprecipitation have been performed on 10.000 cells using the µChIPmentation Kit for Histones (Cat. No. C01011011) and 24 UDI for Tagmented libraries – Set I (Cat. No. Cat. No. C01011034) using K562 cells. The Diagenode antibodies targeting H3K4me3 (Cat. No. C15410003) and rabbit IgG (Cat. No. C15410206) have been used. </small></p>
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<div class="row">
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<p><strong>ATAC-seq</strong>, Assay for<span> </span><strong>T</strong>ransposase-<strong>A</strong>ccessible<span> </span><strong>C</strong>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the<span> </span><strong>transposase Tn5</strong><span> </span>which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing. ATAC-seq methods allow you to:</p>
<ul>
<li> Gain insight into gene regulation and understand open chromatin signatures</li>
<li> Determine nucleosome positions at single nucleotide resolution</li>
<li> Uncover transcription factor (TF) occupancy</li>
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<p>Diagenode’s<span> </span><b>ATAC-</b><b>seq</b><b><span> </span>kit<span> </span></b>is based on a highly validated protocol optimized for<span> </span><b>50,000<span> </span></b><b>cells</b><b><span> </span>per<span> </span></b><b>reaction</b>. The kit includes the reagents for cell lysis and nuclei extraction, tagmentation and DNA purification as well as for library amplification. The <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">primer indexes for multiplexing</a> are not included in the kit and must be purchased separately.</p>
<h4><span style="font-weight: 400;">ATAC-seq kit features:</span></h4>
<ul>
<li><b>Cell<span> </span></b><b>requirement</b><b>:<span> </span></b><b>50,000<span> </span></b><b>cells /<span> </span></b><b>rxn</b></li>
<li><b>Robust protocol<span> </span></b>with<span> </span><b>high reproducibility<span> </span></b>between replicates and repetitive experiments</li>
<li><strong>Easy</strong><span> </span>and<span> </span><b>efficient DNA capture<span> </span></b>after the tagmentation reaction using Diagenode`s MicroChIP DiaPure columns (included)</li>
<li>Additional qPCR step to determine the number of cycles needed for library amplification: </li>
<ul type="”square”">
<li><b>Avoids<span> </span></b><b>over-amplification</b></li>
<li>Allows adaptation/flexibility for<span> </span><b>more challenging samples<span> </span></b>to succeed with library prep.</li>
<li>Gives<span> </span><strong>early indication</strong><span> </span>if the experiment does not work (no qPCR amplification)</li>
</ul>
</ul>
<p>Looking for ATAC-seq on tissue? Please, go to: <a href="https://www.diagenode.com/en/p/ATAC-seq-package-tissue-C01080006">ATAC-seq package for tissue</a></p>',
'label1' => 'Method overview',
'info1' => '<p><strong>ATAC-seq</strong>, <strong>A</strong>ssay for <strong>T</strong>ransposase-<strong>A</strong>ccessible <strong>C</strong>hromatin, followed by next generation sequencing, is a key technology to easily identify the <strong>open regions of the chromatin.</strong> The protocol consists of <strong>3 steps</strong>: <strong>nuclei preparation</strong>, <strong>tagmentation</strong> and <strong>library amplification</strong>. First, the cells undergo the lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq.png" alt="ATAC-seq kit workflow" width="600px" caption="false" /></p>',
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<p><strong>Figure 1.</strong>Representative Bioanalyzer profile of an ATAC-seq library prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig2.png" alt="Diagenode ATAC-seq kit " caption="false" width="951" height="148" /></p>
<p><strong>Figure 2.</strong> Main ATAC-seq alignment and peak calling statistics of 3 replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells. (Mapping efficiency: Percentage of non-mitochondrial reads that mapped to the reference genome. Uniquely mapped ratio: Proportion of mapped reads that map to only one location on the reference genome (hg19). Peaks: Number of peaks (open chromatin regions) identified by MACS2 for each sample. FRiP - Fraction of reads in peaks: Percentage of reads in peaks, with respect to the number of uniquely mapped reads. Sequencing was realized in paired-end mode 50 base pairs (PE50) on an Illumina NovaSeq6000.)</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3a.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3b.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><strong>Figure 3</strong> Sequencing profiles of ATAC-seq library (3 replicates) prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig4.png" alt=" open chromatin regions" caption="false" width="383" height="739" /></p>
<p><strong>Figure 4. </strong><br /> Heatmap around TSS of three ATAC-seq replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>',
'label3' => 'Additional solutions for ATAC-seq kit',
'info3' => '<p><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></p>
<p>Magnetic rack:<span> </span><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"><span> </span>0.2 ml – Cat. No. B04000001</a></p>
<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"><span> </span>(Tn5 transposase)<span> </span></a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">loaded</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"><span> </span>Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns</a><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">, Cat. No. C03040001</a></li>
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/atacseq-kit-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
</ul>
<p><strong>Features:</strong></p>
<ul>
<li>Complete solution for the ATAC-seq workflow</li>
<li>Highly efficient nuclei extraction from tissue</li>
<li>Validated on many mammalian tissues</li>
<li>Compatible with Illumina sequencing platforms</li>
</ul>
<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><br /> <img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq-tissue.png" alt="workflow" style="display: block; margin-left: auto; margin-right: auto;" width="600px" /></p>
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'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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'label3' => 'Additional solutions for ATAC-seq for tissue',
'info3' => '<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
</ul>
<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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'description' => '<p><span>Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the </span><i>Pcsk9</i><span><span> </span>gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.</span></p>',
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'name' => 'Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2',
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'description' => '<p><span>We developed an </span><i>in vivo</i><span><span> </span>hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3′ UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in a slow but progressive, competitive expansion of GFP</span><sup>+</sup><span><span> </span>PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the levels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. Whole-exome sequencing did not show significant differences in the genomic instability indices between tHGMGA2/GFP mice and untreated control mice. Auto-expansion by tHMGA2 was validated in humanized mice. This is the first demonstration that simple injections of mobilization drugs and HDAd vectors can trigger auto-expansion of<span> </span></span><i>in vivo</i><span><span> </span>transduced HSCs resulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.</span></p>',
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'description' => '<p id="p-2">Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads.</p>
<p id="p-3">Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers.</p>
<p id="p-4">SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human diseases.</p>',
'date' => '2024-08-08',
'pmid' => 'https://www.biorxiv.org/content/10.1101/2024.08.08.607188v1',
'doi' => 'https://doi.org/10.1101/2024.08.08.607188',
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'description' => '<p><span>Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different </span><i>Escherichia coli</i><span><span> </span>strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.</span></p>',
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'pmid' => 'https://www.nature.com/articles/s41598-024-57537-6',
'doi' => 'https://doi.org/10.1038/s41598-024-57537-6',
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'name' => 'Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma',
'authors' => 'Kim IK et al.',
'description' => '<p><span>Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (</span><i>Irf6</i><span>), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.</span></p>',
'date' => '2024-02-20',
'pmid' => 'https://www.nature.com/articles/s41467-024-46048-7',
'doi' => 'https://doi.org/10.1038/s41467-024-46048-7',
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'name' => 'CompDuplex: Accurate detection of somatic mutations by duplex-seq with comprehensive genome coverage',
'authors' => 'Muchun Niu et al.',
'description' => '<div class="_dvu6yd">
<section class="_fz2017">
<section class="_protocols-io-draft _lw40b6">
<section class="_protocols-io-draft-app _protocols-io-draft-app-reader _awu6vp">
<section class="_protocols-io-draft-app-editor protocols-io-draft-app-editor-reader">
<div class="DraftEditor-root">
<div class="DraftEditor-editorContainer">
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<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="c6pdl-0-0">
<div data-offset-key="c6pdl-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="c6pdl-0-0"><span data-text="true">Somatic mutations continuously accumulate in the human genome, posing vulnerabilities towards aging and increased risk of various diseases. However, accurate detection of somatic mutations at the whole genome scale is still challenging. By tagging and independently sequencing the two complementary strands of DNA, the recent development of duplex-sequencing methods has greatly improved the detection accuracy, however, the limited genome coverage and the compromised compatibility with existing sequencing platforms have constrained the broad applications of these methods.</span></span></div>
</div>
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="cbilg-0-0">
<div data-offset-key="cbilg-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="cbilg-0-0"> </span></div>
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<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="bud8b-0-0">
<div data-offset-key="bud8b-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="bud8b-0-0"><span data-text="true">To overcome these technical challenges, here we developed a duplex sequencing method with comprehensive genome coverage, which we refer to as CompDuplex-seq. The streamlined chemistry of CompDuplex assay allows efficient generation of libraries readily compatible with standard Illumina 2x150 paired-end sequencing. In addition, we validated the accuracy of somatic mutation calling and comprehensive genome coverage of CompDuplex by profiling a single-cell expanded clone. To summarize, CompDuplex chemistry supports genome-wide coverage while maintaining high accuracy, which we believe will facilitate the whole genome characterization of somatic mosaicism in various biological systems.</span></span></div>
</div>
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</section>',
'date' => '2024-01-25',
'pmid' => 'https://www.protocols.io/view/compduplex-accurate-detection-of-somatic-mutations-kxygx3x4og8j/v1',
'doi' => 'dx.doi.org/10.17504/protocols.io.kxygx3x4og8j/v1',
'modified' => '2024-01-29 10:08:44',
'created' => '2024-01-29 10:08:44',
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[maximum depth reached]
)
),
(int) 6 => array(
'id' => '4893',
'name' => 'Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans',
'authors' => 'Yuanqing Feng et al.',
'description' => '<p><span>Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for </span><i>MFSD12</i><span>,<span> </span></span><i>HMG20B</i><span>,<span> </span></span><i>OCA2</i><span>,<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span>,<span> </span></span><i>TRPS1</i><span>,<span> </span></span><i>BLOC1S6</i><span><span> </span>and<span> </span></span><i>CYB561A3</i><span><span> </span>that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an<span> </span></span><i>OCA2</i><span><span> </span>enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span><span> </span>and<span> </span></span><i>TRPS1</i><span>, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified<span> </span></span><i>CYB561A3</i><span><span> </span>as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.</span></p>',
'date' => '2024-01-10',
'pmid' => 'https://www.nature.com/articles/s41588-023-01626-1',
'doi' => 'https://doi.org/10.1038/s41588-023-01626-1',
'modified' => '2024-01-15 10:24:09',
'created' => '2024-01-15 10:24:09',
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[maximum depth reached]
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),
(int) 7 => array(
'id' => '4879',
'name' => 'A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo',
'authors' => 'Bestas B. et al.',
'description' => '<div id="Abs1" lang="en" class="tsec sec">
<div>
<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
</div>
<div class="sec"><strong class="kwd-title">Subject terms:<span> </span></strong><span class="kwd-text">Genetic engineering, Gene therapy, CRISPR-Cas9 genome editing</span></div>
</div>
<div id="Abs2" lang="en" class="tsec sec"></div>',
'date' => '2023-09-06',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10482872/',
'doi' => '10.1038/s41467-023-41240-7',
'modified' => '2023-11-10 15:00:50',
'created' => '2023-11-10 15:00:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '4869',
'name' => 'Combined Analysis of mRNA Expression and Open Chromatin in Microglia',
'authors' => 'Scholz R.et al.',
'description' => '<p><span>The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.</span></p>',
'date' => '2023-08-29',
'pmid' => 'https://link.springer.com/protocol/10.1007/978-1-0716-3437-0_35',
'doi' => '10.1007/978-1-0716-3437-0_35',
'modified' => '2023-08-31 11:25:45',
'created' => '2023-08-31 11:18:53',
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[maximum depth reached]
)
),
(int) 9 => array(
'id' => '4877',
'name' => 'Volumetric imaging of an intact organism by a distributed molecular network',
'authors' => 'Nianchao Qian and Joshua A Weinstein',
'description' => '<p><span>Lymphatic, nervous, and tumoral tissues, among others, exhibit physiology that emerges from three-dimensional interactions between genetically unique cells. A technology capable of volumetrically imaging transcriptomes, genotypes, and morphologies in a single de novo measurement would therefore provide a critical view into the biological complexity of living systems. Here we achieve this by extending DNA microscopy, an imaging modality that encodes a spatio-genetic map of a specimen via a massive distributed network of DNA molecules inside it, to three dimensions and multiple length scales in developing zebrafish embryos.</span></p>',
'date' => '2023-08-14',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/37645933/',
'doi' => '10.1101/2023.08.11.553025',
'modified' => '2023-11-10 14:45:12',
'created' => '2023-11-10 14:45:12',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '4781',
'name' => 'Spatial epigenome-transcriptome co-profiling of mammalian tissues.',
'authors' => 'Zhang D. et al.',
'description' => '<p>Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36922587',
'doi' => '10.1038/s41586-023-05795-1',
'modified' => '2023-06-13 09:17:26',
'created' => '2023-05-05 12:34:24',
'ProductsPublication' => array(
[maximum depth reached]
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),
(int) 11 => array(
'id' => '4757',
'name' => 'Analyzing genomic and epigenetic profiles in single cells by hybridtransposase (scGET-seq).',
'authors' => 'Cittaro D. et al.',
'description' => '<p>scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022) and de Pretis and Cittaro (2022)..</p>',
'date' => '2023-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37000619',
'doi' => '10.1016/j.xpro.2023.102176',
'modified' => '2023-04-17 09:04:55',
'created' => '2023-04-14 13:41:22',
'ProductsPublication' => array(
[maximum depth reached]
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),
(int) 12 => array(
'id' => '4548',
'name' => 'Imaging Chromatin Accessibility by Assay ofTransposase-Accessible Chromatin with Visualization.',
'authors' => 'Miyanari Yusuke',
'description' => '<p>Chromatin accessibility is one of the fundamental structures regulating genome functions including transcription and DNA repair. Recent technological advantages to analyze chromatin accessibility begun to explore the dynamics of local chromatin structures. Here I describe protocols for Assay of Transposase-Accessible Chromatin with Visualization (ATAC-see), which allows us to analyze subnuclear localization of accessible chromatin and quantify accessible chromatin at single-cell level.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36173568',
'doi' => '10.1007/978-1-0716-2724-2_7',
'modified' => '2022-11-24 10:28:08',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '4654',
'name' => 'Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing',
'authors' => 'Li Haikuo and Humphreys Benjamin D.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.</p>',
'date' => '2022-12-01',
'pmid' => 'https://doi.org/10.1016%2Fj.xpro.2022.101904',
'doi' => '10.1016/j.xpro.2022.101904',
'modified' => '2023-08-01 14:23:49',
'created' => '2023-02-21 09:59:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 14 => array(
'id' => '4546',
'name' => 'Optimized single-nucleus transcriptional profiling by combinatorialindexing.',
'authors' => 'Martin Beth K et al.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36261634',
'doi' => '10.1038/s41596-022-00752-0',
'modified' => '2022-11-24 10:26:25',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 15 => array(
'id' => '4412',
'name' => 'Spatial profiling of chromatin accessibility in mouse and human tissues',
'authors' => 'Yanxiang Deng et al.',
'description' => '<p><span>Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Perkel, J. M. Starfish enterprise: finding RNA patterns in single cells. Nature 572, 549–551 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR1" id="ref-link-section-d163865808e834">1</a></sup><span>. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. Y. & Zhuang, X. W. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR2" id="ref-link-section-d163865808e838">2</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+. Nature 568, 235–239 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR3" id="ref-link-section-d163865808e838_1">3</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR4" id="ref-link-section-d163865808e838_2">4</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e841">5</a></sup><span>, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR6" id="ref-link-section-d163865808e845">6</a></sup><span><span> </span>and microfluidic deterministic barcoding</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e849">5</a></sup><span>. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.</span></p>',
'date' => '2022-08-05',
'pmid' => 'https://www.nature.com/articles/s41586-022-05094-1',
'doi' => 'https://doi.org/10.1038/s41586-022-05094-1',
'modified' => '2022-08-23 11:54:39',
'created' => '2022-08-23 11:54:39',
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[maximum depth reached]
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(int) 16 => array(
'id' => '4389',
'name' => 'Spatially resolved epigenome-transcriptome co-profiling of mammalian tissues at the cellular level',
'authors' => 'Fan Rong et al.',
'description' => '<p>Emerging spatial technologies including spatial transcriptomics and spatial epigenomics are becoming powerful tools for profiling cellular states in the tissue context. However, current methods capture only one layer of omics information at a time precluding the possibility to examine the mechanistic relationship across the cental dogma of molecular biology. Here, we present two spatial multi-omics technologies for spatially resolved genome-wide joint mapping of epigenome and transcriptome by coprofiling chromatin accessibility and gene expression (spatial-ATAC-RNA-seq) or histone modification and gene expression (spatial-CUT\&Tag-RNA-seq) on the same tissue section at a resolution near single cells. They were applied to embryonic and neonatal mouse brain as well as adult human brain to map how epigenetic states or modifications regulate cell type and dynamics in tissue. Although distinct tissue features were identified by either spatial epigenome or spatial transcriptome alone with high concordance, we observed their differential roles in defining cell states. In general, epigenetic state proceeds the development of transcriptional phenotype in relation to epigenetic lineage priming. We also observed high expression canonical markers such as PROX1 in the granular cell layer of the human hippocampus showed low chromatin accessibility that corresponded to a low level of RNA turnover rate, highlighting a divergent need for open chromatin or transcription to control cell identity and dynamics. Spatial epigenome-transcriptome co-profiling is a highly informative tool to study the mechanism of gene expression regulation in tissue and may enable a wide range of applications in life science and biomedical research.</p>',
'date' => '2022-06-13',
'pmid' => 'https://www.researchsquare.com/article/rs-1728747/v1',
'doi' => '10.21203/rs.3.rs-1728747/v1',
'modified' => '2022-08-11 15:20:45',
'created' => '2022-08-11 12:14:50',
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[maximum depth reached]
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(int) 17 => array(
'id' => '4101',
'name' => 'Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues',
'authors' => 'Liguo Zhang et al.',
'description' => '<p>Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3′ end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.</p>',
'date' => '2021-05-25',
'pmid' => 'https://www.pnas.org/content/118/21/e2105968118',
'doi' => 'https://doi.org/10.1073/pnas.2105968118',
'modified' => '2021-06-24 09:49:41',
'created' => '2021-06-24 09:45:16',
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'id' => '4641',
'name' => 'T-RHEX-RNAseq – A tagmentation-based, rRNA blocked, randomhexamer primed RNAseq method for generating stranded RNAseq librariesdirectly from very low numbers of lysed cells',
'authors' => 'Gustafsson Charlotte et al.',
'description' => '<p>Background: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts – remains challenging. Results: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. Conclusion: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.1101%2F2022.10.20.513000',
'doi' => '10.1101/2022.10.20.513000',
'modified' => '2023-03-13 10:57:55',
'created' => '2023-02-21 09:59:46',
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'description' => '<p>We experienced strong purity and activity differences between in-house produced Tn5 batches and<strong> switched to buying Tn5 from Diagenode</strong><span> </span>with<span> </span><strong><u>higher activity and small batch effects</u></strong><span> </span>only.</p>',
'author' => 'Rebekka Scholz et al. Combined Analysis of mRNA Expression and Open Chromatin in Microglia. Methods Mol Biol. 2024;2713:543-571. ',
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'description' => '<p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p>',
'author' => 'Julia Liz Touza, AstraZeneca Gothenburg, Sweden',
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<p>Diagenode Tagmentase is a hyperactive Tn5 transposase with the potential to enhance epigenetic studies. Its ability to cut DNA and insert sequences of interest in one step makes it the perfect companion for Next-Generation Sequencing experiments using powerful technologies such as ATAC-seq, ChIPmentation, CHANGE-seq and other. The enzyme is not loaded with DNA oligos, providing flexibility of application. To ensure optimal results the concentration may be adjusted with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-dilution-buffer">Tagmentase Dilution Buffer</a> (Cat. No. C01070011), available separately.</p>
<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<div><span>Protein Molecular weight: 53.3 kDa</span></div>
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<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
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<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p><cite>Julia Liz Touza, AstraZeneca Gothenburg, Sweden</cite></blockquote>
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<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
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<li>Highly efficient nuclei extraction from tissue</li>
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<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
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'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
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<p>Other supplies:</p>
<ul>
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<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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<div><span>Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
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'authors' => 'Gustafsson Charlotte et al.',
'description' => '<p>Background: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts – remains challenging. Results: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. Conclusion: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.</p>',
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
</ul>
<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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'info2' => '<p><strong>Tagmentase (Tn5 transposase) - unloaded</strong></p>
<div><span style="font-family: inherit;">Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong> Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions:</strong> Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer: </strong>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p>Diagenode <strong>Tagmentation Buffer (2x)</strong> is the recommended reagent to perform any tagmentation reactions. It can be used in combination with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase)</a> on DNA or chromatin samples, as half of the total volume reaction like in ATAC-seq protocol.</p>
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'info1' => '<p><span style="text-decoration: underline;">ATAC-seq experiments: </span></p>
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<li>After cell lysis and nuclei isolation, the nuclei pellets can be incubated with the following mix for 1 reaction:</li>
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<td style="width: 326px;">Tagmentation Buffer (2x)</td>
<td style="width: 114px; padding-left: 30px;">25 µl</td>
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<td style="width: 114px; padding-left: 30px;">2.5 µl</td>
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<td style="width: 326px;"><span>Digitonin 1%</span></td>
<td style="width: 114px; padding-left: 30px;">0.5 µl</td>
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<td style="width: 326px;">Tween20 10%</td>
<td style="width: 114px; padding-left: 30px;">0.5 µl</td>
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<td style="width: 114px; padding-left: 30px;">16.5 µl</td>
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<td style="width: 114px; padding-left: 30px;"> 5 µl</td>
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<p><em>* The number of nuclei per reaction will depend on the ATAC-seq experimental design. Successful tagmentation with the proposed protocol has been performed on 50,000 nuclei per reaction. </em></p>
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<li>The reaction is then incubated 30 minutes at 37°C.</li>
<li>The tagmentation reaction can then be stopped by addition of 250 µl of DNA Binding buffer from Diagenode MicroChIP DiaPure Columns (Cat. No. C03040001).</li>
<li>The tagmented libraries can then be purified using the MicroChIP DiaPure Columns (Cat. No. C03040001), and amplified.</li>
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<p>The <strong>24 UDI for tagmented libraries</strong> includes 24 primer pairs for unique dual-indexing allowing the multiplexing of up to <b>24 samples </b>for sequencing on Illumina platforms. These UDI are designed and validated to be used with <a href="https://www.diagenode.com/en/p/uchipmentation-for-histones-24-rxns">μChIPmentation for Histones</a> (Cat. No. C01011011), <a href="https://www.diagenode.com/en/p/chipmentation-kit-for-histones">ChIPmentation Kit for Histones</a> (Cat. No. C01011009), <a href="https://www.diagenode.com/en/p/tag-kit-for-chipmentation-24">TAG Kit for ChIPmentation</a> (Cat. No. C01011030), <a href="https://www.diagenode.com/en/categories/atac-seq">ATAC-seq Kit</a> (Cat. No. C01080002). The 24 UDI for tagmented libraries are compatible with other <b>tagmentation</b><b>-based library preparation </b>protocols, such as <a href="https://www.diagenode.com/en/categories/cutandtag">CUT&Tag</a> technologies.</p>
<p>3 sets of UDI for tagmented libraries are available:</p>
<p><strong>24 UDI for tagmented libraries - Set I</strong><br /> <a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set2">24 UDI for tagmented libraries - Set II</a><br /><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set3" target="_blank">24 UDI for tagmented libraries - Set III</a><br /><br /></p>
<p><span>Each set can be used for library multiplexing up to 24. All sets can be used simultaneously for library multiplexing up to 72.</span></p>
<p>Features:</p>
<ul>
<li>Multiplexing: <b>up to 72 samples </b>(using all 3 sets simultaneously)<b><br /></b></li>
<li>Allow for <b>identification of index hopping</b></li>
<li>Compatibility: <b>tagmentation</b><b>-based library preparation protocols</b></li>
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'info1' => '<p>The <b>24 UDI (Unique dual indexes) for </b><b>tagmented</b><b> libraries – Set I </b>is compatible with any <b>tagmentation</b><b>-based library preparation </b>protocols, such as <strong>ChIPmentation</strong>, <b>ATAC-seq</b> or <b>CUT&Tag</b> technologies.</p>
<p>The <b>24 UDI for </b><b>tagmented</b><b> libraries </b>provides combinations of barcodes where each barcode is uniquely attributed to one sample. This is a great tool to identify mistakes during index sequencing. A phenomenon, known as index hopping, can lead to misattribution of some reads to the wrong sample. This is particularly frequent with the NovaSeq6000, and thus the use of Unique Dual Indexing (UDI) is highly recommended when using this sequencer.</p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/UDI-for-tagmented-fig1.png" /></center>
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<p><small><strong>Figure 1. Sequencing profiles of µChIPmentation libraries generated with 24 UDI for Tagmented libraries</strong> Chromatin preparation and immunoprecipitation have been performed on 10.000 cells using the µChIPmentation Kit for Histones (Cat. No. C01011011) and 24 UDI for Tagmented libraries – Set I (Cat. No. Cat. No. C01011034) using K562 cells. The Diagenode antibodies targeting H3K4me3 (Cat. No. C15410003) and rabbit IgG (Cat. No. C15410206) have been used. </small></p>
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'name' => 'ATAC-seq kit',
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<div class="row">
<div class="small-12 medium-8 large-8 columns"><br />
<p><strong>ATAC-seq</strong>, Assay for<span> </span><strong>T</strong>ransposase-<strong>A</strong>ccessible<span> </span><strong>C</strong>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the<span> </span><strong>transposase Tn5</strong><span> </span>which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing. ATAC-seq methods allow you to:</p>
<ul>
<li> Gain insight into gene regulation and understand open chromatin signatures</li>
<li> Determine nucleosome positions at single nucleotide resolution</li>
<li> Uncover transcription factor (TF) occupancy</li>
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<p>Diagenode’s<span> </span><b>ATAC-</b><b>seq</b><b><span> </span>kit<span> </span></b>is based on a highly validated protocol optimized for<span> </span><b>50,000<span> </span></b><b>cells</b><b><span> </span>per<span> </span></b><b>reaction</b>. The kit includes the reagents for cell lysis and nuclei extraction, tagmentation and DNA purification as well as for library amplification. The <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">primer indexes for multiplexing</a> are not included in the kit and must be purchased separately.</p>
<h4><span style="font-weight: 400;">ATAC-seq kit features:</span></h4>
<ul>
<li><b>Cell<span> </span></b><b>requirement</b><b>:<span> </span></b><b>50,000<span> </span></b><b>cells /<span> </span></b><b>rxn</b></li>
<li><b>Robust protocol<span> </span></b>with<span> </span><b>high reproducibility<span> </span></b>between replicates and repetitive experiments</li>
<li><strong>Easy</strong><span> </span>and<span> </span><b>efficient DNA capture<span> </span></b>after the tagmentation reaction using Diagenode`s MicroChIP DiaPure columns (included)</li>
<li>Additional qPCR step to determine the number of cycles needed for library amplification: </li>
<ul type="”square”">
<li><b>Avoids<span> </span></b><b>over-amplification</b></li>
<li>Allows adaptation/flexibility for<span> </span><b>more challenging samples<span> </span></b>to succeed with library prep.</li>
<li>Gives<span> </span><strong>early indication</strong><span> </span>if the experiment does not work (no qPCR amplification)</li>
</ul>
</ul>
<p>Looking for ATAC-seq on tissue? Please, go to: <a href="https://www.diagenode.com/en/p/ATAC-seq-package-tissue-C01080006">ATAC-seq package for tissue</a></p>',
'label1' => 'Method overview',
'info1' => '<p><strong>ATAC-seq</strong>, <strong>A</strong>ssay for <strong>T</strong>ransposase-<strong>A</strong>ccessible <strong>C</strong>hromatin, followed by next generation sequencing, is a key technology to easily identify the <strong>open regions of the chromatin.</strong> The protocol consists of <strong>3 steps</strong>: <strong>nuclei preparation</strong>, <strong>tagmentation</strong> and <strong>library amplification</strong>. First, the cells undergo the lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq.png" alt="ATAC-seq kit workflow" width="600px" caption="false" /></p>',
'label2' => 'Example of results',
'info2' => '<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig1.png" alt="library prepared with the Diagenode ATAC-seq kit " width="500px" caption="false" /></p>
<p><strong>Figure 1.</strong>Representative Bioanalyzer profile of an ATAC-seq library prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig2.png" alt="Diagenode ATAC-seq kit " caption="false" width="951" height="148" /></p>
<p><strong>Figure 2.</strong> Main ATAC-seq alignment and peak calling statistics of 3 replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells. (Mapping efficiency: Percentage of non-mitochondrial reads that mapped to the reference genome. Uniquely mapped ratio: Proportion of mapped reads that map to only one location on the reference genome (hg19). Peaks: Number of peaks (open chromatin regions) identified by MACS2 for each sample. FRiP - Fraction of reads in peaks: Percentage of reads in peaks, with respect to the number of uniquely mapped reads. Sequencing was realized in paired-end mode 50 base pairs (PE50) on an Illumina NovaSeq6000.)</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3a.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3b.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><strong>Figure 3</strong> Sequencing profiles of ATAC-seq library (3 replicates) prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig4.png" alt=" open chromatin regions" caption="false" width="383" height="739" /></p>
<p><strong>Figure 4. </strong><br /> Heatmap around TSS of three ATAC-seq replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>',
'label3' => 'Additional solutions for ATAC-seq kit',
'info3' => '<p><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></p>
<p>Magnetic rack:<span> </span><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"><span> </span>0.2 ml – Cat. No. B04000001</a></p>
<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"><span> </span>(Tn5 transposase)<span> </span></a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">loaded</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"><span> </span>Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns</a><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">, Cat. No. C03040001</a></li>
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'meta_description' => 'Diagenode’s ATAC-seq kit provides a robust protocol for assessing genome-wide chromatin accessibility',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/atacseq-kit-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
</ul>
<p><strong>Features:</strong></p>
<ul>
<li>Complete solution for the ATAC-seq workflow</li>
<li>Highly efficient nuclei extraction from tissue</li>
<li>Validated on many mammalian tissues</li>
<li>Compatible with Illumina sequencing platforms</li>
</ul>
<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><br /> <img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq-tissue.png" alt="workflow" style="display: block; margin-left: auto; margin-right: auto;" width="600px" /></p>
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'label2' => 'Example of results',
'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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'label3' => 'Additional solutions for ATAC-seq for tissue',
'info3' => '<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
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<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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'description' => '<p><span>Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the </span><i>Pcsk9</i><span><span> </span>gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.</span></p>',
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'name' => 'Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2',
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'description' => '<p><span>We developed an </span><i>in vivo</i><span><span> </span>hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3′ UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in a slow but progressive, competitive expansion of GFP</span><sup>+</sup><span><span> </span>PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the levels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. Whole-exome sequencing did not show significant differences in the genomic instability indices between tHGMGA2/GFP mice and untreated control mice. Auto-expansion by tHMGA2 was validated in humanized mice. This is the first demonstration that simple injections of mobilization drugs and HDAd vectors can trigger auto-expansion of<span> </span></span><i>in vivo</i><span><span> </span>transduced HSCs resulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.</span></p>',
'date' => '2024-09-12',
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'description' => '<p id="p-2">Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads.</p>
<p id="p-3">Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers.</p>
<p id="p-4">SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human diseases.</p>',
'date' => '2024-08-08',
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'doi' => 'https://doi.org/10.1101/2024.08.08.607188',
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'description' => '<p><span>Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different </span><i>Escherichia coli</i><span><span> </span>strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.</span></p>',
'date' => '2024-03-21',
'pmid' => 'https://www.nature.com/articles/s41598-024-57537-6',
'doi' => 'https://doi.org/10.1038/s41598-024-57537-6',
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'name' => 'Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma',
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'description' => '<p><span>Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (</span><i>Irf6</i><span>), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.</span></p>',
'date' => '2024-02-20',
'pmid' => 'https://www.nature.com/articles/s41467-024-46048-7',
'doi' => 'https://doi.org/10.1038/s41467-024-46048-7',
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'description' => '<div class="_dvu6yd">
<section class="_fz2017">
<section class="_protocols-io-draft _lw40b6">
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<div data-offset-key="c6pdl-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="c6pdl-0-0"><span data-text="true">Somatic mutations continuously accumulate in the human genome, posing vulnerabilities towards aging and increased risk of various diseases. However, accurate detection of somatic mutations at the whole genome scale is still challenging. By tagging and independently sequencing the two complementary strands of DNA, the recent development of duplex-sequencing methods has greatly improved the detection accuracy, however, the limited genome coverage and the compromised compatibility with existing sequencing platforms have constrained the broad applications of these methods.</span></span></div>
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<div data-offset-key="bud8b-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="bud8b-0-0"><span data-text="true">To overcome these technical challenges, here we developed a duplex sequencing method with comprehensive genome coverage, which we refer to as CompDuplex-seq. The streamlined chemistry of CompDuplex assay allows efficient generation of libraries readily compatible with standard Illumina 2x150 paired-end sequencing. In addition, we validated the accuracy of somatic mutation calling and comprehensive genome coverage of CompDuplex by profiling a single-cell expanded clone. To summarize, CompDuplex chemistry supports genome-wide coverage while maintaining high accuracy, which we believe will facilitate the whole genome characterization of somatic mosaicism in various biological systems.</span></span></div>
</div>
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</section>',
'date' => '2024-01-25',
'pmid' => 'https://www.protocols.io/view/compduplex-accurate-detection-of-somatic-mutations-kxygx3x4og8j/v1',
'doi' => 'dx.doi.org/10.17504/protocols.io.kxygx3x4og8j/v1',
'modified' => '2024-01-29 10:08:44',
'created' => '2024-01-29 10:08:44',
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[maximum depth reached]
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(int) 6 => array(
'id' => '4893',
'name' => 'Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans',
'authors' => 'Yuanqing Feng et al.',
'description' => '<p><span>Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for </span><i>MFSD12</i><span>,<span> </span></span><i>HMG20B</i><span>,<span> </span></span><i>OCA2</i><span>,<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span>,<span> </span></span><i>TRPS1</i><span>,<span> </span></span><i>BLOC1S6</i><span><span> </span>and<span> </span></span><i>CYB561A3</i><span><span> </span>that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an<span> </span></span><i>OCA2</i><span><span> </span>enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span><span> </span>and<span> </span></span><i>TRPS1</i><span>, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified<span> </span></span><i>CYB561A3</i><span><span> </span>as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.</span></p>',
'date' => '2024-01-10',
'pmid' => 'https://www.nature.com/articles/s41588-023-01626-1',
'doi' => 'https://doi.org/10.1038/s41588-023-01626-1',
'modified' => '2024-01-15 10:24:09',
'created' => '2024-01-15 10:24:09',
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(int) 7 => array(
'id' => '4879',
'name' => 'A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo',
'authors' => 'Bestas B. et al.',
'description' => '<div id="Abs1" lang="en" class="tsec sec">
<div>
<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
</div>
<div class="sec"><strong class="kwd-title">Subject terms:<span> </span></strong><span class="kwd-text">Genetic engineering, Gene therapy, CRISPR-Cas9 genome editing</span></div>
</div>
<div id="Abs2" lang="en" class="tsec sec"></div>',
'date' => '2023-09-06',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10482872/',
'doi' => '10.1038/s41467-023-41240-7',
'modified' => '2023-11-10 15:00:50',
'created' => '2023-11-10 15:00:50',
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(int) 8 => array(
'id' => '4869',
'name' => 'Combined Analysis of mRNA Expression and Open Chromatin in Microglia',
'authors' => 'Scholz R.et al.',
'description' => '<p><span>The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.</span></p>',
'date' => '2023-08-29',
'pmid' => 'https://link.springer.com/protocol/10.1007/978-1-0716-3437-0_35',
'doi' => '10.1007/978-1-0716-3437-0_35',
'modified' => '2023-08-31 11:25:45',
'created' => '2023-08-31 11:18:53',
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(int) 9 => array(
'id' => '4877',
'name' => 'Volumetric imaging of an intact organism by a distributed molecular network',
'authors' => 'Nianchao Qian and Joshua A Weinstein',
'description' => '<p><span>Lymphatic, nervous, and tumoral tissues, among others, exhibit physiology that emerges from three-dimensional interactions between genetically unique cells. A technology capable of volumetrically imaging transcriptomes, genotypes, and morphologies in a single de novo measurement would therefore provide a critical view into the biological complexity of living systems. Here we achieve this by extending DNA microscopy, an imaging modality that encodes a spatio-genetic map of a specimen via a massive distributed network of DNA molecules inside it, to three dimensions and multiple length scales in developing zebrafish embryos.</span></p>',
'date' => '2023-08-14',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/37645933/',
'doi' => '10.1101/2023.08.11.553025',
'modified' => '2023-11-10 14:45:12',
'created' => '2023-11-10 14:45:12',
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(int) 10 => array(
'id' => '4781',
'name' => 'Spatial epigenome-transcriptome co-profiling of mammalian tissues.',
'authors' => 'Zhang D. et al.',
'description' => '<p>Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36922587',
'doi' => '10.1038/s41586-023-05795-1',
'modified' => '2023-06-13 09:17:26',
'created' => '2023-05-05 12:34:24',
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(int) 11 => array(
'id' => '4757',
'name' => 'Analyzing genomic and epigenetic profiles in single cells by hybridtransposase (scGET-seq).',
'authors' => 'Cittaro D. et al.',
'description' => '<p>scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022) and de Pretis and Cittaro (2022)..</p>',
'date' => '2023-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37000619',
'doi' => '10.1016/j.xpro.2023.102176',
'modified' => '2023-04-17 09:04:55',
'created' => '2023-04-14 13:41:22',
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(int) 12 => array(
'id' => '4548',
'name' => 'Imaging Chromatin Accessibility by Assay ofTransposase-Accessible Chromatin with Visualization.',
'authors' => 'Miyanari Yusuke',
'description' => '<p>Chromatin accessibility is one of the fundamental structures regulating genome functions including transcription and DNA repair. Recent technological advantages to analyze chromatin accessibility begun to explore the dynamics of local chromatin structures. Here I describe protocols for Assay of Transposase-Accessible Chromatin with Visualization (ATAC-see), which allows us to analyze subnuclear localization of accessible chromatin and quantify accessible chromatin at single-cell level.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36173568',
'doi' => '10.1007/978-1-0716-2724-2_7',
'modified' => '2022-11-24 10:28:08',
'created' => '2022-11-24 08:49:52',
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[maximum depth reached]
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(int) 13 => array(
'id' => '4654',
'name' => 'Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing',
'authors' => 'Li Haikuo and Humphreys Benjamin D.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.</p>',
'date' => '2022-12-01',
'pmid' => 'https://doi.org/10.1016%2Fj.xpro.2022.101904',
'doi' => '10.1016/j.xpro.2022.101904',
'modified' => '2023-08-01 14:23:49',
'created' => '2023-02-21 09:59:46',
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[maximum depth reached]
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(int) 14 => array(
'id' => '4546',
'name' => 'Optimized single-nucleus transcriptional profiling by combinatorialindexing.',
'authors' => 'Martin Beth K et al.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36261634',
'doi' => '10.1038/s41596-022-00752-0',
'modified' => '2022-11-24 10:26:25',
'created' => '2022-11-24 08:49:52',
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[maximum depth reached]
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(int) 15 => array(
'id' => '4412',
'name' => 'Spatial profiling of chromatin accessibility in mouse and human tissues',
'authors' => 'Yanxiang Deng et al.',
'description' => '<p><span>Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Perkel, J. M. Starfish enterprise: finding RNA patterns in single cells. Nature 572, 549–551 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR1" id="ref-link-section-d163865808e834">1</a></sup><span>. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. Y. & Zhuang, X. W. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR2" id="ref-link-section-d163865808e838">2</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+. Nature 568, 235–239 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR3" id="ref-link-section-d163865808e838_1">3</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR4" id="ref-link-section-d163865808e838_2">4</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e841">5</a></sup><span>, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR6" id="ref-link-section-d163865808e845">6</a></sup><span><span> </span>and microfluidic deterministic barcoding</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e849">5</a></sup><span>. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.</span></p>',
'date' => '2022-08-05',
'pmid' => 'https://www.nature.com/articles/s41586-022-05094-1',
'doi' => 'https://doi.org/10.1038/s41586-022-05094-1',
'modified' => '2022-08-23 11:54:39',
'created' => '2022-08-23 11:54:39',
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(int) 16 => array(
'id' => '4389',
'name' => 'Spatially resolved epigenome-transcriptome co-profiling of mammalian tissues at the cellular level',
'authors' => 'Fan Rong et al.',
'description' => '<p>Emerging spatial technologies including spatial transcriptomics and spatial epigenomics are becoming powerful tools for profiling cellular states in the tissue context. However, current methods capture only one layer of omics information at a time precluding the possibility to examine the mechanistic relationship across the cental dogma of molecular biology. Here, we present two spatial multi-omics technologies for spatially resolved genome-wide joint mapping of epigenome and transcriptome by coprofiling chromatin accessibility and gene expression (spatial-ATAC-RNA-seq) or histone modification and gene expression (spatial-CUT\&Tag-RNA-seq) on the same tissue section at a resolution near single cells. They were applied to embryonic and neonatal mouse brain as well as adult human brain to map how epigenetic states or modifications regulate cell type and dynamics in tissue. Although distinct tissue features were identified by either spatial epigenome or spatial transcriptome alone with high concordance, we observed their differential roles in defining cell states. In general, epigenetic state proceeds the development of transcriptional phenotype in relation to epigenetic lineage priming. We also observed high expression canonical markers such as PROX1 in the granular cell layer of the human hippocampus showed low chromatin accessibility that corresponded to a low level of RNA turnover rate, highlighting a divergent need for open chromatin or transcription to control cell identity and dynamics. Spatial epigenome-transcriptome co-profiling is a highly informative tool to study the mechanism of gene expression regulation in tissue and may enable a wide range of applications in life science and biomedical research.</p>',
'date' => '2022-06-13',
'pmid' => 'https://www.researchsquare.com/article/rs-1728747/v1',
'doi' => '10.21203/rs.3.rs-1728747/v1',
'modified' => '2022-08-11 15:20:45',
'created' => '2022-08-11 12:14:50',
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(int) 17 => array(
'id' => '4101',
'name' => 'Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues',
'authors' => 'Liguo Zhang et al.',
'description' => '<p>Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3′ end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.</p>',
'date' => '2021-05-25',
'pmid' => 'https://www.pnas.org/content/118/21/e2105968118',
'doi' => 'https://doi.org/10.1073/pnas.2105968118',
'modified' => '2021-06-24 09:49:41',
'created' => '2021-06-24 09:45:16',
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'id' => '4641',
'name' => 'T-RHEX-RNAseq – A tagmentation-based, rRNA blocked, randomhexamer primed RNAseq method for generating stranded RNAseq librariesdirectly from very low numbers of lysed cells',
'authors' => 'Gustafsson Charlotte et al.',
'description' => '<p>Background: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts – remains challenging. Results: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. Conclusion: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.1101%2F2022.10.20.513000',
'doi' => '10.1101/2022.10.20.513000',
'modified' => '2023-03-13 10:57:55',
'created' => '2023-02-21 09:59:46',
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'description' => '<p>We experienced strong purity and activity differences between in-house produced Tn5 batches and<strong> switched to buying Tn5 from Diagenode</strong><span> </span>with<span> </span><strong><u>higher activity and small batch effects</u></strong><span> </span>only.</p>',
'author' => 'Rebekka Scholz et al. Combined Analysis of mRNA Expression and Open Chromatin in Microglia. Methods Mol Biol. 2024;2713:543-571. ',
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'description' => '<p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p>',
'author' => 'Julia Liz Touza, AstraZeneca Gothenburg, Sweden',
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<p>Diagenode Tagmentase is a hyperactive Tn5 transposase with the potential to enhance epigenetic studies. Its ability to cut DNA and insert sequences of interest in one step makes it the perfect companion for Next-Generation Sequencing experiments using powerful technologies such as ATAC-seq, ChIPmentation, CHANGE-seq and other. The enzyme is not loaded with DNA oligos, providing flexibility of application. To ensure optimal results the concentration may be adjusted with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-dilution-buffer">Tagmentase Dilution Buffer</a> (Cat. No. C01070011), available separately.</p>
<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
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<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
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<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
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'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
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<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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'info2' => '<p><strong>Tagmentase (Tn5 transposase) - unloaded</strong></p>
<div><span>Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage:</strong><span> </span>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
</ul>
<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><img alt="Tn5 transposase" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure-1a.jpg" style="display: block; margin-left: auto; margin-right: auto;" width="653" height="282" /></p>
<p><img alt="Tagmentase Tn5 transposase" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure-1b.jpg" style="display: block; margin-left: auto; margin-right: auto;" width="645" height="278" /></p>
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<div class="small-12 medium-12 large-12 columns"><center><img alt="Tn5 transposase perfect for NGS" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure2.jpg" width="754" height="492" /></center></div>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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'info2' => '<p><strong>Tagmentase (Tn5 transposase) - unloaded</strong></p>
<div><span style="font-family: inherit;">Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong> Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions:</strong> Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer: </strong>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p>Diagenode <strong>Tagmentation Buffer (2x)</strong> is the recommended reagent to perform any tagmentation reactions. It can be used in combination with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase)</a> on DNA or chromatin samples, as half of the total volume reaction like in ATAC-seq protocol.</p>
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<li>After cell lysis and nuclei isolation, the nuclei pellets can be incubated with the following mix for 1 reaction:</li>
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<td style="width: 326px;">Tagmentation Buffer (2x)</td>
<td style="width: 114px; padding-left: 30px;">25 µl</td>
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<td style="width: 114px; padding-left: 30px;">2.5 µl</td>
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<td style="width: 326px;"><span>Digitonin 1%</span></td>
<td style="width: 114px; padding-left: 30px;">0.5 µl</td>
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<td style="width: 326px;">Tween20 10%</td>
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<td style="width: 114px; padding-left: 30px;"> 5 µl</td>
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<p><em>* The number of nuclei per reaction will depend on the ATAC-seq experimental design. Successful tagmentation with the proposed protocol has been performed on 50,000 nuclei per reaction. </em></p>
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<li>The reaction is then incubated 30 minutes at 37°C.</li>
<li>The tagmentation reaction can then be stopped by addition of 250 µl of DNA Binding buffer from Diagenode MicroChIP DiaPure Columns (Cat. No. C03040001).</li>
<li>The tagmented libraries can then be purified using the MicroChIP DiaPure Columns (Cat. No. C03040001), and amplified.</li>
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<p>The <strong>24 UDI for tagmented libraries</strong> includes 24 primer pairs for unique dual-indexing allowing the multiplexing of up to <b>24 samples </b>for sequencing on Illumina platforms. These UDI are designed and validated to be used with <a href="https://www.diagenode.com/en/p/uchipmentation-for-histones-24-rxns">μChIPmentation for Histones</a> (Cat. No. C01011011), <a href="https://www.diagenode.com/en/p/chipmentation-kit-for-histones">ChIPmentation Kit for Histones</a> (Cat. No. C01011009), <a href="https://www.diagenode.com/en/p/tag-kit-for-chipmentation-24">TAG Kit for ChIPmentation</a> (Cat. No. C01011030), <a href="https://www.diagenode.com/en/categories/atac-seq">ATAC-seq Kit</a> (Cat. No. C01080002). The 24 UDI for tagmented libraries are compatible with other <b>tagmentation</b><b>-based library preparation </b>protocols, such as <a href="https://www.diagenode.com/en/categories/cutandtag">CUT&Tag</a> technologies.</p>
<p>3 sets of UDI for tagmented libraries are available:</p>
<p><strong>24 UDI for tagmented libraries - Set I</strong><br /> <a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set2">24 UDI for tagmented libraries - Set II</a><br /><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set3" target="_blank">24 UDI for tagmented libraries - Set III</a><br /><br /></p>
<p><span>Each set can be used for library multiplexing up to 24. All sets can be used simultaneously for library multiplexing up to 72.</span></p>
<p>Features:</p>
<ul>
<li>Multiplexing: <b>up to 72 samples </b>(using all 3 sets simultaneously)<b><br /></b></li>
<li>Allow for <b>identification of index hopping</b></li>
<li>Compatibility: <b>tagmentation</b><b>-based library preparation protocols</b></li>
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<p>The <b>24 UDI for </b><b>tagmented</b><b> libraries </b>provides combinations of barcodes where each barcode is uniquely attributed to one sample. This is a great tool to identify mistakes during index sequencing. A phenomenon, known as index hopping, can lead to misattribution of some reads to the wrong sample. This is particularly frequent with the NovaSeq6000, and thus the use of Unique Dual Indexing (UDI) is highly recommended when using this sequencer.</p>
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<center><img src="https://www.diagenode.com/img/product/kits/UDI-for-tagmented-fig1.png" /></center>
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<p><small><strong>Figure 1. Sequencing profiles of µChIPmentation libraries generated with 24 UDI for Tagmented libraries</strong> Chromatin preparation and immunoprecipitation have been performed on 10.000 cells using the µChIPmentation Kit for Histones (Cat. No. C01011011) and 24 UDI for Tagmented libraries – Set I (Cat. No. Cat. No. C01011034) using K562 cells. The Diagenode antibodies targeting H3K4me3 (Cat. No. C15410003) and rabbit IgG (Cat. No. C15410206) have been used. </small></p>
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<div class="row">
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<p><strong>ATAC-seq</strong>, Assay for<span> </span><strong>T</strong>ransposase-<strong>A</strong>ccessible<span> </span><strong>C</strong>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the<span> </span><strong>transposase Tn5</strong><span> </span>which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing. ATAC-seq methods allow you to:</p>
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<li> Gain insight into gene regulation and understand open chromatin signatures</li>
<li> Determine nucleosome positions at single nucleotide resolution</li>
<li> Uncover transcription factor (TF) occupancy</li>
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<p>Diagenode’s<span> </span><b>ATAC-</b><b>seq</b><b><span> </span>kit<span> </span></b>is based on a highly validated protocol optimized for<span> </span><b>50,000<span> </span></b><b>cells</b><b><span> </span>per<span> </span></b><b>reaction</b>. The kit includes the reagents for cell lysis and nuclei extraction, tagmentation and DNA purification as well as for library amplification. The <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">primer indexes for multiplexing</a> are not included in the kit and must be purchased separately.</p>
<h4><span style="font-weight: 400;">ATAC-seq kit features:</span></h4>
<ul>
<li><b>Cell<span> </span></b><b>requirement</b><b>:<span> </span></b><b>50,000<span> </span></b><b>cells /<span> </span></b><b>rxn</b></li>
<li><b>Robust protocol<span> </span></b>with<span> </span><b>high reproducibility<span> </span></b>between replicates and repetitive experiments</li>
<li><strong>Easy</strong><span> </span>and<span> </span><b>efficient DNA capture<span> </span></b>after the tagmentation reaction using Diagenode`s MicroChIP DiaPure columns (included)</li>
<li>Additional qPCR step to determine the number of cycles needed for library amplification: </li>
<ul type="”square”">
<li><b>Avoids<span> </span></b><b>over-amplification</b></li>
<li>Allows adaptation/flexibility for<span> </span><b>more challenging samples<span> </span></b>to succeed with library prep.</li>
<li>Gives<span> </span><strong>early indication</strong><span> </span>if the experiment does not work (no qPCR amplification)</li>
</ul>
</ul>
<p>Looking for ATAC-seq on tissue? Please, go to: <a href="https://www.diagenode.com/en/p/ATAC-seq-package-tissue-C01080006">ATAC-seq package for tissue</a></p>',
'label1' => 'Method overview',
'info1' => '<p><strong>ATAC-seq</strong>, <strong>A</strong>ssay for <strong>T</strong>ransposase-<strong>A</strong>ccessible <strong>C</strong>hromatin, followed by next generation sequencing, is a key technology to easily identify the <strong>open regions of the chromatin.</strong> The protocol consists of <strong>3 steps</strong>: <strong>nuclei preparation</strong>, <strong>tagmentation</strong> and <strong>library amplification</strong>. First, the cells undergo the lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq.png" alt="ATAC-seq kit workflow" width="600px" caption="false" /></p>',
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<p><strong>Figure 1.</strong>Representative Bioanalyzer profile of an ATAC-seq library prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig2.png" alt="Diagenode ATAC-seq kit " caption="false" width="951" height="148" /></p>
<p><strong>Figure 2.</strong> Main ATAC-seq alignment and peak calling statistics of 3 replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells. (Mapping efficiency: Percentage of non-mitochondrial reads that mapped to the reference genome. Uniquely mapped ratio: Proportion of mapped reads that map to only one location on the reference genome (hg19). Peaks: Number of peaks (open chromatin regions) identified by MACS2 for each sample. FRiP - Fraction of reads in peaks: Percentage of reads in peaks, with respect to the number of uniquely mapped reads. Sequencing was realized in paired-end mode 50 base pairs (PE50) on an Illumina NovaSeq6000.)</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3a.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3b.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><strong>Figure 3</strong> Sequencing profiles of ATAC-seq library (3 replicates) prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig4.png" alt=" open chromatin regions" caption="false" width="383" height="739" /></p>
<p><strong>Figure 4. </strong><br /> Heatmap around TSS of three ATAC-seq replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>',
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'info3' => '<p><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></p>
<p>Magnetic rack:<span> </span><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"><span> </span>0.2 ml – Cat. No. B04000001</a></p>
<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"><span> </span>(Tn5 transposase)<span> </span></a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">loaded</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"><span> </span>Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns</a><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">, Cat. No. C03040001</a></li>
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'meta_description' => 'Diagenode’s ATAC-seq kit provides a robust protocol for assessing genome-wide chromatin accessibility',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/atacseq-kit-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
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<p><strong>Features:</strong></p>
<ul>
<li>Complete solution for the ATAC-seq workflow</li>
<li>Highly efficient nuclei extraction from tissue</li>
<li>Validated on many mammalian tissues</li>
<li>Compatible with Illumina sequencing platforms</li>
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<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><br /> <img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq-tissue.png" alt="workflow" style="display: block; margin-left: auto; margin-right: auto;" width="600px" /></p>
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'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
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<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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'description' => '<p id="p-2">Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads.</p>
<p id="p-3">Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers.</p>
<p id="p-4">SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human diseases.</p>',
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'name' => 'Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS)',
'authors' => 'Kyono Y. et al.',
'description' => '<p><span>Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different </span><i>Escherichia coli</i><span><span> </span>strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.</span></p>',
'date' => '2024-03-21',
'pmid' => 'https://www.nature.com/articles/s41598-024-57537-6',
'doi' => 'https://doi.org/10.1038/s41598-024-57537-6',
'modified' => '2024-04-10 16:29:00',
'created' => '2024-04-10 16:29:00',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '4916',
'name' => 'Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma',
'authors' => 'Kim IK et al.',
'description' => '<p><span>Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (</span><i>Irf6</i><span>), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.</span></p>',
'date' => '2024-02-20',
'pmid' => 'https://www.nature.com/articles/s41467-024-46048-7',
'doi' => 'https://doi.org/10.1038/s41467-024-46048-7',
'modified' => '2024-02-26 13:39:36',
'created' => '2024-02-26 13:39:36',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 5 => array(
'id' => '4897',
'name' => 'CompDuplex: Accurate detection of somatic mutations by duplex-seq with comprehensive genome coverage',
'authors' => 'Muchun Niu et al.',
'description' => '<div class="_dvu6yd">
<section class="_fz2017">
<section class="_protocols-io-draft _lw40b6">
<section class="_protocols-io-draft-app _protocols-io-draft-app-reader _awu6vp">
<section class="_protocols-io-draft-app-editor protocols-io-draft-app-editor-reader">
<div class="DraftEditor-root">
<div class="DraftEditor-editorContainer">
<div aria-label="" class="public-DraftEditor-content" contenteditable="false" spellcheck="false">
<div data-contents="true">
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="c6pdl-0-0">
<div data-offset-key="c6pdl-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="c6pdl-0-0"><span data-text="true">Somatic mutations continuously accumulate in the human genome, posing vulnerabilities towards aging and increased risk of various diseases. However, accurate detection of somatic mutations at the whole genome scale is still challenging. By tagging and independently sequencing the two complementary strands of DNA, the recent development of duplex-sequencing methods has greatly improved the detection accuracy, however, the limited genome coverage and the compromised compatibility with existing sequencing platforms have constrained the broad applications of these methods.</span></span></div>
</div>
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="cbilg-0-0">
<div data-offset-key="cbilg-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="cbilg-0-0"> </span></div>
</div>
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="bud8b-0-0">
<div data-offset-key="bud8b-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="bud8b-0-0"><span data-text="true">To overcome these technical challenges, here we developed a duplex sequencing method with comprehensive genome coverage, which we refer to as CompDuplex-seq. The streamlined chemistry of CompDuplex assay allows efficient generation of libraries readily compatible with standard Illumina 2x150 paired-end sequencing. In addition, we validated the accuracy of somatic mutation calling and comprehensive genome coverage of CompDuplex by profiling a single-cell expanded clone. To summarize, CompDuplex chemistry supports genome-wide coverage while maintaining high accuracy, which we believe will facilitate the whole genome characterization of somatic mosaicism in various biological systems.</span></span></div>
</div>
</div>
</div>
</div>
</div>
<span id="placeholder-desc-draft-abstract"></span></section>
</section>
</section>
</section>
</div>
<section class="_e296pg">
<div id="step-sticky-section" class="_j60wwa">
<div class="_1oxfq56"></div>
<div class="_wcbn92"></div>
</div>
</section>',
'date' => '2024-01-25',
'pmid' => 'https://www.protocols.io/view/compduplex-accurate-detection-of-somatic-mutations-kxygx3x4og8j/v1',
'doi' => 'dx.doi.org/10.17504/protocols.io.kxygx3x4og8j/v1',
'modified' => '2024-01-29 10:08:44',
'created' => '2024-01-29 10:08:44',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '4893',
'name' => 'Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans',
'authors' => 'Yuanqing Feng et al.',
'description' => '<p><span>Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for </span><i>MFSD12</i><span>,<span> </span></span><i>HMG20B</i><span>,<span> </span></span><i>OCA2</i><span>,<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span>,<span> </span></span><i>TRPS1</i><span>,<span> </span></span><i>BLOC1S6</i><span><span> </span>and<span> </span></span><i>CYB561A3</i><span><span> </span>that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an<span> </span></span><i>OCA2</i><span><span> </span>enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span><span> </span>and<span> </span></span><i>TRPS1</i><span>, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified<span> </span></span><i>CYB561A3</i><span><span> </span>as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.</span></p>',
'date' => '2024-01-10',
'pmid' => 'https://www.nature.com/articles/s41588-023-01626-1',
'doi' => 'https://doi.org/10.1038/s41588-023-01626-1',
'modified' => '2024-01-15 10:24:09',
'created' => '2024-01-15 10:24:09',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '4879',
'name' => 'A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo',
'authors' => 'Bestas B. et al.',
'description' => '<div id="Abs1" lang="en" class="tsec sec">
<div>
<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
</div>
<div class="sec"><strong class="kwd-title">Subject terms:<span> </span></strong><span class="kwd-text">Genetic engineering, Gene therapy, CRISPR-Cas9 genome editing</span></div>
</div>
<div id="Abs2" lang="en" class="tsec sec"></div>',
'date' => '2023-09-06',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10482872/',
'doi' => '10.1038/s41467-023-41240-7',
'modified' => '2023-11-10 15:00:50',
'created' => '2023-11-10 15:00:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '4869',
'name' => 'Combined Analysis of mRNA Expression and Open Chromatin in Microglia',
'authors' => 'Scholz R.et al.',
'description' => '<p><span>The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.</span></p>',
'date' => '2023-08-29',
'pmid' => 'https://link.springer.com/protocol/10.1007/978-1-0716-3437-0_35',
'doi' => '10.1007/978-1-0716-3437-0_35',
'modified' => '2023-08-31 11:25:45',
'created' => '2023-08-31 11:18:53',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '4877',
'name' => 'Volumetric imaging of an intact organism by a distributed molecular network',
'authors' => 'Nianchao Qian and Joshua A Weinstein',
'description' => '<p><span>Lymphatic, nervous, and tumoral tissues, among others, exhibit physiology that emerges from three-dimensional interactions between genetically unique cells. A technology capable of volumetrically imaging transcriptomes, genotypes, and morphologies in a single de novo measurement would therefore provide a critical view into the biological complexity of living systems. Here we achieve this by extending DNA microscopy, an imaging modality that encodes a spatio-genetic map of a specimen via a massive distributed network of DNA molecules inside it, to three dimensions and multiple length scales in developing zebrafish embryos.</span></p>',
'date' => '2023-08-14',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/37645933/',
'doi' => '10.1101/2023.08.11.553025',
'modified' => '2023-11-10 14:45:12',
'created' => '2023-11-10 14:45:12',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '4781',
'name' => 'Spatial epigenome-transcriptome co-profiling of mammalian tissues.',
'authors' => 'Zhang D. et al.',
'description' => '<p>Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36922587',
'doi' => '10.1038/s41586-023-05795-1',
'modified' => '2023-06-13 09:17:26',
'created' => '2023-05-05 12:34:24',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 11 => array(
'id' => '4757',
'name' => 'Analyzing genomic and epigenetic profiles in single cells by hybridtransposase (scGET-seq).',
'authors' => 'Cittaro D. et al.',
'description' => '<p>scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022) and de Pretis and Cittaro (2022)..</p>',
'date' => '2023-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37000619',
'doi' => '10.1016/j.xpro.2023.102176',
'modified' => '2023-04-17 09:04:55',
'created' => '2023-04-14 13:41:22',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 12 => array(
'id' => '4548',
'name' => 'Imaging Chromatin Accessibility by Assay ofTransposase-Accessible Chromatin with Visualization.',
'authors' => 'Miyanari Yusuke',
'description' => '<p>Chromatin accessibility is one of the fundamental structures regulating genome functions including transcription and DNA repair. Recent technological advantages to analyze chromatin accessibility begun to explore the dynamics of local chromatin structures. Here I describe protocols for Assay of Transposase-Accessible Chromatin with Visualization (ATAC-see), which allows us to analyze subnuclear localization of accessible chromatin and quantify accessible chromatin at single-cell level.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36173568',
'doi' => '10.1007/978-1-0716-2724-2_7',
'modified' => '2022-11-24 10:28:08',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '4654',
'name' => 'Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing',
'authors' => 'Li Haikuo and Humphreys Benjamin D.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.</p>',
'date' => '2022-12-01',
'pmid' => 'https://doi.org/10.1016%2Fj.xpro.2022.101904',
'doi' => '10.1016/j.xpro.2022.101904',
'modified' => '2023-08-01 14:23:49',
'created' => '2023-02-21 09:59:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 14 => array(
'id' => '4546',
'name' => 'Optimized single-nucleus transcriptional profiling by combinatorialindexing.',
'authors' => 'Martin Beth K et al.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36261634',
'doi' => '10.1038/s41596-022-00752-0',
'modified' => '2022-11-24 10:26:25',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 15 => array(
'id' => '4412',
'name' => 'Spatial profiling of chromatin accessibility in mouse and human tissues',
'authors' => 'Yanxiang Deng et al.',
'description' => '<p><span>Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Perkel, J. M. Starfish enterprise: finding RNA patterns in single cells. Nature 572, 549–551 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR1" id="ref-link-section-d163865808e834">1</a></sup><span>. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. Y. & Zhuang, X. W. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR2" id="ref-link-section-d163865808e838">2</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+. Nature 568, 235–239 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR3" id="ref-link-section-d163865808e838_1">3</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR4" id="ref-link-section-d163865808e838_2">4</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e841">5</a></sup><span>, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR6" id="ref-link-section-d163865808e845">6</a></sup><span><span> </span>and microfluidic deterministic barcoding</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e849">5</a></sup><span>. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.</span></p>',
'date' => '2022-08-05',
'pmid' => 'https://www.nature.com/articles/s41586-022-05094-1',
'doi' => 'https://doi.org/10.1038/s41586-022-05094-1',
'modified' => '2022-08-23 11:54:39',
'created' => '2022-08-23 11:54:39',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 16 => array(
'id' => '4389',
'name' => 'Spatially resolved epigenome-transcriptome co-profiling of mammalian tissues at the cellular level',
'authors' => 'Fan Rong et al.',
'description' => '<p>Emerging spatial technologies including spatial transcriptomics and spatial epigenomics are becoming powerful tools for profiling cellular states in the tissue context. However, current methods capture only one layer of omics information at a time precluding the possibility to examine the mechanistic relationship across the cental dogma of molecular biology. Here, we present two spatial multi-omics technologies for spatially resolved genome-wide joint mapping of epigenome and transcriptome by coprofiling chromatin accessibility and gene expression (spatial-ATAC-RNA-seq) or histone modification and gene expression (spatial-CUT\&Tag-RNA-seq) on the same tissue section at a resolution near single cells. They were applied to embryonic and neonatal mouse brain as well as adult human brain to map how epigenetic states or modifications regulate cell type and dynamics in tissue. Although distinct tissue features were identified by either spatial epigenome or spatial transcriptome alone with high concordance, we observed their differential roles in defining cell states. In general, epigenetic state proceeds the development of transcriptional phenotype in relation to epigenetic lineage priming. We also observed high expression canonical markers such as PROX1 in the granular cell layer of the human hippocampus showed low chromatin accessibility that corresponded to a low level of RNA turnover rate, highlighting a divergent need for open chromatin or transcription to control cell identity and dynamics. Spatial epigenome-transcriptome co-profiling is a highly informative tool to study the mechanism of gene expression regulation in tissue and may enable a wide range of applications in life science and biomedical research.</p>',
'date' => '2022-06-13',
'pmid' => 'https://www.researchsquare.com/article/rs-1728747/v1',
'doi' => '10.21203/rs.3.rs-1728747/v1',
'modified' => '2022-08-11 15:20:45',
'created' => '2022-08-11 12:14:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 17 => array(
'id' => '4101',
'name' => 'Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues',
'authors' => 'Liguo Zhang et al.',
'description' => '<p>Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3′ end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.</p>',
'date' => '2021-05-25',
'pmid' => 'https://www.pnas.org/content/118/21/e2105968118',
'doi' => 'https://doi.org/10.1073/pnas.2105968118',
'modified' => '2021-06-24 09:49:41',
'created' => '2021-06-24 09:45:16',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 18 => array(
'id' => '4641',
'name' => 'T-RHEX-RNAseq – A tagmentation-based, rRNA blocked, randomhexamer primed RNAseq method for generating stranded RNAseq librariesdirectly from very low numbers of lysed cells',
'authors' => 'Gustafsson Charlotte et al.',
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<p>Diagenode Tagmentase is a hyperactive Tn5 transposase with the potential to enhance epigenetic studies. Its ability to cut DNA and insert sequences of interest in one step makes it the perfect companion for Next-Generation Sequencing experiments using powerful technologies such as ATAC-seq, ChIPmentation, CHANGE-seq and other. The enzyme is not loaded with DNA oligos, providing flexibility of application. To ensure optimal results the concentration may be adjusted with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-dilution-buffer">Tagmentase Dilution Buffer</a> (Cat. No. C01070011), available separately.</p>
<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
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<p><strong>Properties & Usage:</strong><span> </span>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<div class="small-12 medium-12 large-12 columns">
<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
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<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p><cite>Julia Liz Touza, AstraZeneca Gothenburg, Sweden</cite></blockquote>
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<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
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<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
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<li>Highly efficient nuclei extraction from tissue</li>
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<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
</ul>
<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
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<p>Diagenode Tagmentase is a hyperactive Tn5 transposase with the potential to enhance epigenetic studies. Its ability to cut DNA and insert sequences of interest in one step makes it the perfect companion for Next-Generation Sequencing experiments using powerful technologies such as ATAC-seq, ChIPmentation, CHANGE-seq and other. The enzyme is not loaded with DNA oligos, providing flexibility of application. To ensure optimal results the concentration may be adjusted with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-dilution-buffer">Tagmentase Dilution Buffer</a> (Cat. No. C01070011), available separately.</p>
<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
</ul>
<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><img alt="Tn5 transposase" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure-1a.jpg" style="display: block; margin-left: auto; margin-right: auto;" width="653" height="282" /></p>
<p><img alt="Tagmentase Tn5 transposase" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure-1b.jpg" style="display: block; margin-left: auto; margin-right: auto;" width="645" height="278" /></p>
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<div><span style="font-family: inherit;">Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong> Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions:</strong> Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer: </strong>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p>Diagenode <strong>Tagmentation Buffer (2x)</strong> is the recommended reagent to perform any tagmentation reactions. It can be used in combination with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase)</a> on DNA or chromatin samples, as half of the total volume reaction like in ATAC-seq protocol.</p>
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<li>After cell lysis and nuclei isolation, the nuclei pellets can be incubated with the following mix for 1 reaction:</li>
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<td style="width: 326px;">Tagmentation Buffer (2x)</td>
<td style="width: 114px; padding-left: 30px;">25 µl</td>
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<td style="width: 326px;"><span>Digitonin 1%</span></td>
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<p><em>* The number of nuclei per reaction will depend on the ATAC-seq experimental design. Successful tagmentation with the proposed protocol has been performed on 50,000 nuclei per reaction. </em></p>
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<li>The reaction is then incubated 30 minutes at 37°C.</li>
<li>The tagmentation reaction can then be stopped by addition of 250 µl of DNA Binding buffer from Diagenode MicroChIP DiaPure Columns (Cat. No. C03040001).</li>
<li>The tagmented libraries can then be purified using the MicroChIP DiaPure Columns (Cat. No. C03040001), and amplified.</li>
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<p>The <strong>24 UDI for tagmented libraries</strong> includes 24 primer pairs for unique dual-indexing allowing the multiplexing of up to <b>24 samples </b>for sequencing on Illumina platforms. These UDI are designed and validated to be used with <a href="https://www.diagenode.com/en/p/uchipmentation-for-histones-24-rxns">μChIPmentation for Histones</a> (Cat. No. C01011011), <a href="https://www.diagenode.com/en/p/chipmentation-kit-for-histones">ChIPmentation Kit for Histones</a> (Cat. No. C01011009), <a href="https://www.diagenode.com/en/p/tag-kit-for-chipmentation-24">TAG Kit for ChIPmentation</a> (Cat. No. C01011030), <a href="https://www.diagenode.com/en/categories/atac-seq">ATAC-seq Kit</a> (Cat. No. C01080002). The 24 UDI for tagmented libraries are compatible with other <b>tagmentation</b><b>-based library preparation </b>protocols, such as <a href="https://www.diagenode.com/en/categories/cutandtag">CUT&Tag</a> technologies.</p>
<p>3 sets of UDI for tagmented libraries are available:</p>
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<li>Allow for <b>identification of index hopping</b></li>
<li>Compatibility: <b>tagmentation</b><b>-based library preparation protocols</b></li>
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<p>The <b>24 UDI for </b><b>tagmented</b><b> libraries </b>provides combinations of barcodes where each barcode is uniquely attributed to one sample. This is a great tool to identify mistakes during index sequencing. A phenomenon, known as index hopping, can lead to misattribution of some reads to the wrong sample. This is particularly frequent with the NovaSeq6000, and thus the use of Unique Dual Indexing (UDI) is highly recommended when using this sequencer.</p>
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<p><small><strong>Figure 1. Sequencing profiles of µChIPmentation libraries generated with 24 UDI for Tagmented libraries</strong> Chromatin preparation and immunoprecipitation have been performed on 10.000 cells using the µChIPmentation Kit for Histones (Cat. No. C01011011) and 24 UDI for Tagmented libraries – Set I (Cat. No. Cat. No. C01011034) using K562 cells. The Diagenode antibodies targeting H3K4me3 (Cat. No. C15410003) and rabbit IgG (Cat. No. C15410206) have been used. </small></p>
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<p><strong>ATAC-seq</strong>, Assay for<span> </span><strong>T</strong>ransposase-<strong>A</strong>ccessible<span> </span><strong>C</strong>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the<span> </span><strong>transposase Tn5</strong><span> </span>which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing. ATAC-seq methods allow you to:</p>
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<p>Diagenode’s<span> </span><b>ATAC-</b><b>seq</b><b><span> </span>kit<span> </span></b>is based on a highly validated protocol optimized for<span> </span><b>50,000<span> </span></b><b>cells</b><b><span> </span>per<span> </span></b><b>reaction</b>. The kit includes the reagents for cell lysis and nuclei extraction, tagmentation and DNA purification as well as for library amplification. The <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">primer indexes for multiplexing</a> are not included in the kit and must be purchased separately.</p>
<h4><span style="font-weight: 400;">ATAC-seq kit features:</span></h4>
<ul>
<li><b>Cell<span> </span></b><b>requirement</b><b>:<span> </span></b><b>50,000<span> </span></b><b>cells /<span> </span></b><b>rxn</b></li>
<li><b>Robust protocol<span> </span></b>with<span> </span><b>high reproducibility<span> </span></b>between replicates and repetitive experiments</li>
<li><strong>Easy</strong><span> </span>and<span> </span><b>efficient DNA capture<span> </span></b>after the tagmentation reaction using Diagenode`s MicroChIP DiaPure columns (included)</li>
<li>Additional qPCR step to determine the number of cycles needed for library amplification: </li>
<ul type="”square”">
<li><b>Avoids<span> </span></b><b>over-amplification</b></li>
<li>Allows adaptation/flexibility for<span> </span><b>more challenging samples<span> </span></b>to succeed with library prep.</li>
<li>Gives<span> </span><strong>early indication</strong><span> </span>if the experiment does not work (no qPCR amplification)</li>
</ul>
</ul>
<p>Looking for ATAC-seq on tissue? Please, go to: <a href="https://www.diagenode.com/en/p/ATAC-seq-package-tissue-C01080006">ATAC-seq package for tissue</a></p>',
'label1' => 'Method overview',
'info1' => '<p><strong>ATAC-seq</strong>, <strong>A</strong>ssay for <strong>T</strong>ransposase-<strong>A</strong>ccessible <strong>C</strong>hromatin, followed by next generation sequencing, is a key technology to easily identify the <strong>open regions of the chromatin.</strong> The protocol consists of <strong>3 steps</strong>: <strong>nuclei preparation</strong>, <strong>tagmentation</strong> and <strong>library amplification</strong>. First, the cells undergo the lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq.png" alt="ATAC-seq kit workflow" width="600px" caption="false" /></p>',
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<p><strong>Figure 1.</strong>Representative Bioanalyzer profile of an ATAC-seq library prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig2.png" alt="Diagenode ATAC-seq kit " caption="false" width="951" height="148" /></p>
<p><strong>Figure 2.</strong> Main ATAC-seq alignment and peak calling statistics of 3 replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells. (Mapping efficiency: Percentage of non-mitochondrial reads that mapped to the reference genome. Uniquely mapped ratio: Proportion of mapped reads that map to only one location on the reference genome (hg19). Peaks: Number of peaks (open chromatin regions) identified by MACS2 for each sample. FRiP - Fraction of reads in peaks: Percentage of reads in peaks, with respect to the number of uniquely mapped reads. Sequencing was realized in paired-end mode 50 base pairs (PE50) on an Illumina NovaSeq6000.)</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3a.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig3b.png" alt="Assay for Transposase-Accessible Chromatin" width="500px" caption="false" /></p>
<p><strong>Figure 3</strong> Sequencing profiles of ATAC-seq library (3 replicates) prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>
<p><img src="https://www.diagenode.com/img/product/kits/atacseq-fig4.png" alt=" open chromatin regions" caption="false" width="383" height="739" /></p>
<p><strong>Figure 4. </strong><br /> Heatmap around TSS of three ATAC-seq replicates prepared with the Diagenode ATAC-seq kit and 24 UDI for tagmented libraries (Cat. No. C01011034) on 50,000 nuclei from K562 cells.</p>',
'label3' => 'Additional solutions for ATAC-seq kit',
'info3' => '<p><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></p>
<p>Magnetic rack:<span> </span><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"><span> </span>0.2 ml – Cat. No. B04000001</a></p>
<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"><span> </span>(Tn5 transposase)<span> </span></a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">loaded</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"><span> </span>Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a><span> </span><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns</a><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">, Cat. No. C03040001</a></li>
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/atacseq-kit-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
</ul>
<p><strong>Features:</strong></p>
<ul>
<li>Complete solution for the ATAC-seq workflow</li>
<li>Highly efficient nuclei extraction from tissue</li>
<li>Validated on many mammalian tissues</li>
<li>Compatible with Illumina sequencing platforms</li>
</ul>
<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
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'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><br /> <img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq-tissue.png" alt="workflow" style="display: block; margin-left: auto; margin-right: auto;" width="600px" /></p>
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'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
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<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
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'id' => '4966',
'name' => 'Detection of genome structural variation in normal cells and tissues by single molecule sequencing',
'authors' => 'Heid J. et al.',
'description' => '<p id="p-2">Detecting somatic mutations in normal cells and tissues is notoriously challenging due to their low abundance, orders of magnitude below the sequencing error rate. While several techniques, such as single-cell and single-molecule sequencing, have been developed to identify somatic mutations, they are insufficient for detecting genomic structural variants (SVs), which have a significantly greater impact than single-nucleotide variants (SNVs). We introduce Single-Molecule Mutation Sequencing for Structural Variants (SMM-SV-seq), a novel method combining Tn5-mediated, chimera-free library preparation with the precision of error-corrected next-generation sequencing (ecNGS). This approach enhances SV detection accuracy without relying on independent supporting sequencing reads.</p>
<p id="p-3">Our validation studies on human primary fibroblasts treated with varying concentrations of the clastogen bleomycin demonstrated a significant, up to tenfold and dose-dependent, increase in deletions and translocations 24 hours post-treatment. Evaluating SMM-SV-seq’s performance against established computational tools for SV detection, such as Manta and DELLY, using a well-characterized human cell line, SMM-SV-seq showed precision and recall rates of 61.9% and 85.8%, respectively, significantly outperforming Manta (10% precision, 23% recall) and DELLY (15% precision, 32% recall). Using SMM-SV-seq, we documented clear, direct evidence of negative selection against structural variants over time. After a single 2 Gy dose of ionizing radiation, SVs in normal human primary fibroblasts peaked at 24 hours post-intervention and then declined to nearly background levels by day six, highlighting the cellular mechanisms that selectively disadvantage cells harboring these mutations. Additionally, SMM-SV-seq revealed that BRCA1-deficient human breast epithelial cells are more susceptible to the mutagenic effects of ionizing radiation compared to BRCA1-proficient isogenic control cells, suggesting a potential molecular mechanism for increased breast cancer risk in BRCA1 mutation carriers.</p>
<p id="p-4">SMM-SV-seq represents a significant advancement in genomic analysis, enabling the accurate detection of somatic structural variants in normal cells and tissues for the first time. This method complements our previously published Single-Molecule Mutation sequencing (SMM-seq), effective for detecting single-nucleotide variants (SNVs) and small insertions and deletions (INDELs). By addressing challenges such as self-ligation in library preparation and leveraging a powerful ecNGS strategy, SMM-SV-seq enhances the robustness of our genomic analysis toolkit. This breakthrough paves the way for new research into genetic variability and mutation processes, offering deeper insights that could advance our understanding of aging, cancer, and other human diseases.</p>',
'date' => '2024-08-08',
'pmid' => 'https://www.biorxiv.org/content/10.1101/2024.08.08.607188v1',
'doi' => 'https://doi.org/10.1101/2024.08.08.607188',
'modified' => '2024-09-02 10:27:20',
'created' => '2024-09-02 10:27:20',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 3 => array(
'id' => '4936',
'name' => 'Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS)',
'authors' => 'Kyono Y. et al.',
'description' => '<p><span>Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different </span><i>Escherichia coli</i><span><span> </span>strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.</span></p>',
'date' => '2024-03-21',
'pmid' => 'https://www.nature.com/articles/s41598-024-57537-6',
'doi' => 'https://doi.org/10.1038/s41598-024-57537-6',
'modified' => '2024-04-10 16:29:00',
'created' => '2024-04-10 16:29:00',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '4916',
'name' => 'Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma',
'authors' => 'Kim IK et al.',
'description' => '<p><span>Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (</span><i>Irf6</i><span>), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.</span></p>',
'date' => '2024-02-20',
'pmid' => 'https://www.nature.com/articles/s41467-024-46048-7',
'doi' => 'https://doi.org/10.1038/s41467-024-46048-7',
'modified' => '2024-02-26 13:39:36',
'created' => '2024-02-26 13:39:36',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 5 => array(
'id' => '4897',
'name' => 'CompDuplex: Accurate detection of somatic mutations by duplex-seq with comprehensive genome coverage',
'authors' => 'Muchun Niu et al.',
'description' => '<div class="_dvu6yd">
<section class="_fz2017">
<section class="_protocols-io-draft _lw40b6">
<section class="_protocols-io-draft-app _protocols-io-draft-app-reader _awu6vp">
<section class="_protocols-io-draft-app-editor protocols-io-draft-app-editor-reader">
<div class="DraftEditor-root">
<div class="DraftEditor-editorContainer">
<div aria-label="" class="public-DraftEditor-content" contenteditable="false" spellcheck="false">
<div data-contents="true">
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="c6pdl-0-0">
<div data-offset-key="c6pdl-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="c6pdl-0-0"><span data-text="true">Somatic mutations continuously accumulate in the human genome, posing vulnerabilities towards aging and increased risk of various diseases. However, accurate detection of somatic mutations at the whole genome scale is still challenging. By tagging and independently sequencing the two complementary strands of DNA, the recent development of duplex-sequencing methods has greatly improved the detection accuracy, however, the limited genome coverage and the compromised compatibility with existing sequencing platforms have constrained the broad applications of these methods.</span></span></div>
</div>
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="cbilg-0-0">
<div data-offset-key="cbilg-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="cbilg-0-0"> </span></div>
</div>
<div class=" align-justify" data-block="true" data-editor="desc-draft-abstract" data-offset-key="bud8b-0-0">
<div data-offset-key="bud8b-0-0" class="public-DraftStyleDefault-block public-DraftStyleDefault-ltr"><span data-offset-key="bud8b-0-0"><span data-text="true">To overcome these technical challenges, here we developed a duplex sequencing method with comprehensive genome coverage, which we refer to as CompDuplex-seq. The streamlined chemistry of CompDuplex assay allows efficient generation of libraries readily compatible with standard Illumina 2x150 paired-end sequencing. In addition, we validated the accuracy of somatic mutation calling and comprehensive genome coverage of CompDuplex by profiling a single-cell expanded clone. To summarize, CompDuplex chemistry supports genome-wide coverage while maintaining high accuracy, which we believe will facilitate the whole genome characterization of somatic mosaicism in various biological systems.</span></span></div>
</div>
</div>
</div>
</div>
</div>
<span id="placeholder-desc-draft-abstract"></span></section>
</section>
</section>
</section>
</div>
<section class="_e296pg">
<div id="step-sticky-section" class="_j60wwa">
<div class="_1oxfq56"></div>
<div class="_wcbn92"></div>
</div>
</section>',
'date' => '2024-01-25',
'pmid' => 'https://www.protocols.io/view/compduplex-accurate-detection-of-somatic-mutations-kxygx3x4og8j/v1',
'doi' => 'dx.doi.org/10.17504/protocols.io.kxygx3x4og8j/v1',
'modified' => '2024-01-29 10:08:44',
'created' => '2024-01-29 10:08:44',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '4893',
'name' => 'Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans',
'authors' => 'Yuanqing Feng et al.',
'description' => '<p><span>Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for </span><i>MFSD12</i><span>,<span> </span></span><i>HMG20B</i><span>,<span> </span></span><i>OCA2</i><span>,<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span>,<span> </span></span><i>TRPS1</i><span>,<span> </span></span><i>BLOC1S6</i><span><span> </span>and<span> </span></span><i>CYB561A3</i><span><span> </span>that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an<span> </span></span><i>OCA2</i><span><span> </span>enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of<span> </span></span><i>MITF</i><span>,<span> </span></span><i>LEF1</i><span><span> </span>and<span> </span></span><i>TRPS1</i><span>, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified<span> </span></span><i>CYB561A3</i><span><span> </span>as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.</span></p>',
'date' => '2024-01-10',
'pmid' => 'https://www.nature.com/articles/s41588-023-01626-1',
'doi' => 'https://doi.org/10.1038/s41588-023-01626-1',
'modified' => '2024-01-15 10:24:09',
'created' => '2024-01-15 10:24:09',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '4879',
'name' => 'A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo',
'authors' => 'Bestas B. et al.',
'description' => '<div id="Abs1" lang="en" class="tsec sec">
<div>
<p id="Par1" class="p p-first-last"><em>Streptococcus pyogenes</em><span> </span>Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized<span> </span><em>PCSK9</em><span> </span>mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from<span> </span><em>Parasutterella secunda</em><span> </span>(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the<span> </span><em>PCSK9</em><span> </span>site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.</p>
</div>
<div class="sec"><strong class="kwd-title">Subject terms:<span> </span></strong><span class="kwd-text">Genetic engineering, Gene therapy, CRISPR-Cas9 genome editing</span></div>
</div>
<div id="Abs2" lang="en" class="tsec sec"></div>',
'date' => '2023-09-06',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10482872/',
'doi' => '10.1038/s41467-023-41240-7',
'modified' => '2023-11-10 15:00:50',
'created' => '2023-11-10 15:00:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '4869',
'name' => 'Combined Analysis of mRNA Expression and Open Chromatin in Microglia',
'authors' => 'Scholz R.et al.',
'description' => '<p><span>The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.</span></p>',
'date' => '2023-08-29',
'pmid' => 'https://link.springer.com/protocol/10.1007/978-1-0716-3437-0_35',
'doi' => '10.1007/978-1-0716-3437-0_35',
'modified' => '2023-08-31 11:25:45',
'created' => '2023-08-31 11:18:53',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '4877',
'name' => 'Volumetric imaging of an intact organism by a distributed molecular network',
'authors' => 'Nianchao Qian and Joshua A Weinstein',
'description' => '<p><span>Lymphatic, nervous, and tumoral tissues, among others, exhibit physiology that emerges from three-dimensional interactions between genetically unique cells. A technology capable of volumetrically imaging transcriptomes, genotypes, and morphologies in a single de novo measurement would therefore provide a critical view into the biological complexity of living systems. Here we achieve this by extending DNA microscopy, an imaging modality that encodes a spatio-genetic map of a specimen via a massive distributed network of DNA molecules inside it, to three dimensions and multiple length scales in developing zebrafish embryos.</span></p>',
'date' => '2023-08-14',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/37645933/',
'doi' => '10.1101/2023.08.11.553025',
'modified' => '2023-11-10 14:45:12',
'created' => '2023-11-10 14:45:12',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '4781',
'name' => 'Spatial epigenome-transcriptome co-profiling of mammalian tissues.',
'authors' => 'Zhang D. et al.',
'description' => '<p>Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36922587',
'doi' => '10.1038/s41586-023-05795-1',
'modified' => '2023-06-13 09:17:26',
'created' => '2023-05-05 12:34:24',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 11 => array(
'id' => '4757',
'name' => 'Analyzing genomic and epigenetic profiles in single cells by hybridtransposase (scGET-seq).',
'authors' => 'Cittaro D. et al.',
'description' => '<p>scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022) and de Pretis and Cittaro (2022)..</p>',
'date' => '2023-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37000619',
'doi' => '10.1016/j.xpro.2023.102176',
'modified' => '2023-04-17 09:04:55',
'created' => '2023-04-14 13:41:22',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 12 => array(
'id' => '4548',
'name' => 'Imaging Chromatin Accessibility by Assay ofTransposase-Accessible Chromatin with Visualization.',
'authors' => 'Miyanari Yusuke',
'description' => '<p>Chromatin accessibility is one of the fundamental structures regulating genome functions including transcription and DNA repair. Recent technological advantages to analyze chromatin accessibility begun to explore the dynamics of local chromatin structures. Here I describe protocols for Assay of Transposase-Accessible Chromatin with Visualization (ATAC-see), which allows us to analyze subnuclear localization of accessible chromatin and quantify accessible chromatin at single-cell level.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36173568',
'doi' => '10.1007/978-1-0716-2724-2_7',
'modified' => '2022-11-24 10:28:08',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '4654',
'name' => 'Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing',
'authors' => 'Li Haikuo and Humphreys Benjamin D.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.</p>',
'date' => '2022-12-01',
'pmid' => 'https://doi.org/10.1016%2Fj.xpro.2022.101904',
'doi' => '10.1016/j.xpro.2022.101904',
'modified' => '2023-08-01 14:23:49',
'created' => '2023-02-21 09:59:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 14 => array(
'id' => '4546',
'name' => 'Optimized single-nucleus transcriptional profiling by combinatorialindexing.',
'authors' => 'Martin Beth K et al.',
'description' => '<p>Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36261634',
'doi' => '10.1038/s41596-022-00752-0',
'modified' => '2022-11-24 10:26:25',
'created' => '2022-11-24 08:49:52',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 15 => array(
'id' => '4412',
'name' => 'Spatial profiling of chromatin accessibility in mouse and human tissues',
'authors' => 'Yanxiang Deng et al.',
'description' => '<p><span>Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Perkel, J. M. Starfish enterprise: finding RNA patterns in single cells. Nature 572, 549–551 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR1" id="ref-link-section-d163865808e834">1</a></sup><span>. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. Y. & Zhuang, X. W. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR2" id="ref-link-section-d163865808e838">2</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+. Nature 568, 235–239 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR3" id="ref-link-section-d163865808e838_1">3</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR4" id="ref-link-section-d163865808e838_2">4</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e841">5</a></sup><span>, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR6" id="ref-link-section-d163865808e845">6</a></sup><span><span> </span>and microfluidic deterministic barcoding</span><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Liu, Y. et al. High-spatial-resolution multi-omics sequencing via deterministic barcoding in tissue. Cell 183, 1665–1681 (2020)." href="https://www.nature.com/articles/s41586-022-05094-1#ref-CR5" id="ref-link-section-d163865808e849">5</a></sup><span>. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.</span></p>',
'date' => '2022-08-05',
'pmid' => 'https://www.nature.com/articles/s41586-022-05094-1',
'doi' => 'https://doi.org/10.1038/s41586-022-05094-1',
'modified' => '2022-08-23 11:54:39',
'created' => '2022-08-23 11:54:39',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 16 => array(
'id' => '4389',
'name' => 'Spatially resolved epigenome-transcriptome co-profiling of mammalian tissues at the cellular level',
'authors' => 'Fan Rong et al.',
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<p>Diagenode Tagmentase is a hyperactive Tn5 transposase with the potential to enhance epigenetic studies. Its ability to cut DNA and insert sequences of interest in one step makes it the perfect companion for Next-Generation Sequencing experiments using powerful technologies such as ATAC-seq, ChIPmentation, CHANGE-seq and other. The enzyme is not loaded with DNA oligos, providing flexibility of application. To ensure optimal results the concentration may be adjusted with Diagenode <a href="https://www.diagenode.com/en/p/tagmentase-dilution-buffer">Tagmentase Dilution Buffer</a> (Cat. No. C01070011), available separately.</p>
<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
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<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may also need:</p>
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<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x-1ml">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
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<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
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<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong> Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions:</strong> Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
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<p><strong>Applications: </strong>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
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<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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<p><a href="https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf" target="_blank">Protocol for transposome assembly</a></p>
<p>Using Diagenode’s Tagmentase (Tn5 transposase) you may need also:</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-1x">Tagmentation Buffer (1x)</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation Buffer (2x)</a></li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for tagmented libraries</a></li>
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<p>Looking for loaded Tagmentase? Please go to <a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase (Tn5 transposase) - loaded</a>.</p>',
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<p><strong>Figure 1: Efficient fragmentation of the lambda DNA after incubation with the Tagmentase</strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with diluted Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). Profiles show the size of lambda DNA before (A) and after treatment with Tagmentase (B).</p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns"><center><img alt="Tn5 transposase perfect for NGS" src="https://www.diagenode.com/img/product/reagents/tagmentase-figure2.jpg" width="754" height="492" /></center></div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>Figure 2: Fragmentation efficiency depending on the amount of Tagmentase </strong><br />For fragmentation, 100 ng of DNA from bacteriophage lambda were incubated with Diagenode Tagmentase (Cat. No. C01070010) and Tagmentation buffer (1x) (Cat. No. C01019042) for 7 min at 55°C. The Tagmentase was previously diluted with the Tagmentase Dilution Buffer (Cat. No.) at ¼ and 1/16 dilutions. The reaction was stopped by addition of SDS (0.2% final concentration). After clean-up using AMPure XP beads (Beckman Coulter) on Diagenode IP-Star robot, the size of the DNA was assessed on Fragment Analyzer (Agilent), using the HS Large Fragment 50kb Kit (Agilent). The migration of the samples shows variations of the size distribution according to the amount of Tagmentase used for the reaction.</p>
</div>
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'info2' => '<p><strong>Tagmentase (Tn5 transposase) - product information</strong></p>
<div><span>Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage: </strong>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>',
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'format' => '500 µl or 1000 µl',
'catalog_number' => 'C01070010-500',
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'sf_code' => 'C01070010-D001-000015',
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'meta_description' => 'Diagenode Tagmentase is a hyperactive Tn5 transposase with the ability to cut DNA and insert sequences of interest into any target DNA in one step. The enzyme is not loaded with DNA oligos, providing flexibility of application.',
'modified' => '2024-12-12 11:59:57',
'created' => '2021-10-20 15:46:20',
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$featured_testimonials = '<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p>We experienced strong purity and activity differences between in-house produced Tn5 batches and<strong> switched to buying Tn5 from Diagenode</strong><span> </span>with<span> </span><strong><u>higher activity and small batch effects</u></strong><span> </span>only.</p><cite>Rebekka Scholz et al. Combined Analysis of mRNA Expression and Open Chromatin in Microglia. Methods Mol Biol. 2024;2713:543-571. </cite></blockquote>
<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p><cite>Julia Liz Touza, AstraZeneca Gothenburg, Sweden</cite></blockquote>
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'description' => '<p><span>We have been using the Hyperactive Tagmentase for 2 years and its performance is outstanding - short operation time and good reproducibility, outmatching the competition. Moreover the interaction with customer representatives is always top-notch - highly efficient and knowledgeable. I can't recommend enough!</span></p>',
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'modified' => '2023-02-08 09:40:13',
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'
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'id' => '3215',
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'name' => 'ATAC-seq package for tissue',
'description' => '<p><a href="https://www.diagenode.com/files/products/kits/atacseq-kit-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><b>ATAC-seq</b>, Assay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology for genome-wide mapping of accessible chromatin. The technology is based on the use of the <b>transposase Tn5</b> which cuts exposed open chromatin and simultaneously ligates adapters for subsequent amplification and sequencing.</p>
<p>The Diagenode’s <b>ATAC-</b><b>seq</b><b> package for tissue </b>has been specifically developted and optimized to generate the ATAC-seq libraries from tissue samples on <b>25 to 100 mg of tissue per </b><b>reaction</b>. The protocol has been validated on many different mammalian tissues (lung, liver, brain, kidney, muscles) and different species (pork, chicken, rat, mice, horse). The package includes the reagents for complete ATAC-seq workflow, including nuclei extraction, library preparation and multiplexing.</p>
<p><strong>Content of the ATAC-seq package for tissues:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004" target="_blank" title="Tissue Nuclei Extraction for ATAC-seq">Tissue<span> </span>Nuclei<span> </span>Extraction for ATAC-seq</a><span> </span>– optimized protocol and reagents for highly efficient nuclei isolation from tissue, preserving the nuclei</li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq<span> </span>kit</a><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns"><span> </span></a>– generation of high quality libraries</li>
<li><a href="https://www.diagenode.com/en/p/24-unique-dual-indexes-for-tagmented-libraries-set1">24 UDI for<span> </span>tagmented<span> </span>libraries*</a><a href="https://www.diagenode.com/en/p/8-unique-dual-indexes-for-tagmented-libraries"><span> </span></a>– efficient multiplexing allowing for index hopping identification and filtering. </li>
</ul>
<p><strong>Features:</strong></p>
<ul>
<li>Complete solution for the ATAC-seq workflow</li>
<li>Highly efficient nuclei extraction from tissue</li>
<li>Validated on many mammalian tissues</li>
<li>Compatible with Illumina sequencing platforms</li>
</ul>
<p>Looking for ATAC-seq for cells? Please go to<span> </span><a href="https://www.diagenode.com/en/p/atac-seq-kit-8rxns">ATAC-seq kit</a>.</p>
<p><em>* For libraries multiplexing, the ATAC-seq package 24 rxns includes the 24 UDI for tagmented libraries kit - set I, Cat. No. C01011034. If needed, higher multiplexing is possible using other sets of <a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries" target="_blank" title="Primer indexes for tagmented libraries">Primer indexes for tagmented libraries</a>, available separately.</em></p>
<p></p>
<p><small><img src="https://icons.iconarchive.com/icons/wikipedia/flags/256/EU-European-Union-Flag-icon.png" alt="" width="45" /> The project GENE-SWitCH leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 817998.<small></small></small></p>',
'label1' => 'Method overview',
'info1' => '<p><b>ATAC-seq</b>, <b>A</b>ssay for <b>T</b>ransposase-<b>A</b>ccessible <b>C</b>hromatin, followed by next generation sequencing, is a key technology to easily identify the <b>open regions of the chromatin.</b> The protocol consists of <b>3 steps</b>: <b>nuclei preparation</b>, <b>tagmentation</b> and <b>library amplification</b>. First, the tissue undergoes lysis, ending with the crude nuclei. Then, the nuclei are incubated with a tagmentase (Tn5 transposase), which cuts the genomic regions associated with open chromatin and inserts the sequencing adaptors. Finally, the generated libraries are amplified and can be used for sequencing. High-throughput sequencing will then detect peaks, in open regions of the chromatin only, giving a map of the chromatin status in the whole genome of the sample.</p>
<p><br /> <img src="https://www.diagenode.com/img/product/kits/workflow-atac-seq-tissue.png" alt="workflow" style="display: block; margin-left: auto; margin-right: auto;" width="600px" /></p>
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'label2' => 'Example of results',
'info2' => '<p>GENE-SWitCH aims to deliver new underpinning knowledge on the functional genomes of two main monogastric farm species (pig and chicken) and to enable immediate translation to the pig and poultry sectors. It is a multi-actor project that will produce new genome information to enable the characterization of genetic and epigenetic determinants of complex traits in these two species. Diagenode, as a principal participant to the project and leading the WP1, developed a new protocol to improve the preparation of ATAC-seq libraries from a variety of snap-frozen tissues. The ATAC-seq protocol combines efficient nuclei extraction procedure validated on 7 different kinds of tissues from 3 developmental stages of the two species and a robust Tagmentation protocol based on Diagenode Tn5 enzyme. The developed ATAC-seq protocol was successfully used to produce 168 ATAC-seq libraries for WP1 and 320 for WP5.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/table1-atacseq-results.png" width="400" /></center>
<p><small><strong>Table 1.</strong> List of validated tissues with Diagenode’s ATAC-seq package for tissue (Cat. No. C01080005/6). The samples were used as part of GENE-SWitCH consortium.</small></p>
<p>A.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2a-atacseq-results.png" width="700" /></center>
<p>B.</p>
<center><img src="https://www.diagenode.com/img/product/kits/atacseq/fig2b-atacseq-results.png" width="700" /></center>
<p><small><strong>Figure 2.</strong> ATAC-seq library profiles generated using the ATAC-seq package for tissue (Cat. No. C01080005/6) from pork’s liver (A) and brain (B). The samples were used as part of GENE-SWitCH consortium.</small></p>
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'label3' => 'Additional solutions for ATAC-seq for tissue',
'info3' => '<p>Additional supplies (included in the kit and available separately):</p>
<ul>
<li><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30">Tagmentase</a><a href="https://www.diagenode.com/en/p/tagmentase-loaded-30"> (Tn5 transposase) loaded, Cat. No. C01070012</a></li>
<li><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x">Tagmentation</a><a href="https://www.diagenode.com/en/p/tagmentation-buffer-2x"> Buffer (2x), Cat. No. C01019043</a></li>
<li><a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">MicroChIP</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">DiaPure</a> <a href="https://www.diagenode.com/en/p/microchip-diapure-columns-50-rxns">columns, Cat. No. C03040001</a></li>
<li><a href="https://www.diagenode.com/en/p/tissue-nuclei-extraction-ATAC-seq-C01080004">Tissue Nuclei Extraction for ATAC-seq, Cat. No. C0108004</a></li>
<li><a href="https://www.diagenode.com/en/p/atac-seq-kit-24rxns">ATAC-seq kit, Cat. No. C01080002</a></li>
</ul>
<p>Other supplies:</p>
<ul>
<li><a href="https://www.diagenode.com/en/categories/primer-indexes-for-tagmented-libraries">Primer indexes for tagmented libraries</a></li>
<li><a href="https://www.diagenode.com/en/p/protease-inhibitor-mix-100-ul">Protease Inhibitor Mix 200X</a></li>
<li>Magnetic rack: <a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit">DiaMag</a><a href="https://www.diagenode.com/en/p/diamag02-magnetic-rack-1-unit"> 0.2 ml – Cat. No. B04000001</a></li>
</ul>
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<div><span>Protein Molecular weight: 53.3 kDa</span></div>
<p>Expressed: in Escherichia coli</p>
<p><strong>Product description:</strong><span> </span>Diagenode Tagmentase – unloaded is a hyperactive Tn5 transposase. The enzyme catalyzes “cut and paste” tagmentation reaction and can be used to insert any target DNA in vitro.</p>
<p><strong>Storage conditions: </strong>Store at -20°C. Guaranteed stable for 6 months from date of receipt when stored properly.</p>
<p><strong>Storage buffer:</strong><span> </span>Supplied in solution containing 50% v/v glycerol.</p>
<p><strong>Properties & Usage:</strong><span> </span>The enzyme should be loaded with appropriate oligonucleotides prior to use. An efficient transposition require that insert DNA have a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each of its ends. The transposome assembly protocol can be found at https://www.diagenode.com/files/protocols/PRO-Transposome-Assembly-V2.pdf Tagmentase is dependent on Mg++ for activity. Avoid chelators, such as EDTA/EGTA, in reaction buffers. The enzyme is active at pH 7.5-8 at 37-55°C. SDS, EDTA/EGTA or heating to 65°C will inactivate the enzyme.</p>
<p><strong>Applications:</strong><span> </span>Tagmentase (Tn5 transposase) – unloaded can be used in a variety of applications including transgenic experiments, barcoding and library construction for second-generation sequencing. Please note that an additional optimization might be required for custom protocols including the enzyme dose- and time-response experiments.</p>
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