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<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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<p><span></span></p>
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<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-96-rxns">C02030042 - Premium Methyl UMI-UDI Adapters - 96 rxns</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
'label2' => 'How it works?',
'info2' => '<center><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/workflow-RRBS_2021.png" width="700px" alt="Premium RRBS kit" caption="false" /></center>
<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
<p><iframe width="320" height="180" src="https://www.youtube.com/embed/CJn3XEAznu0?rel=0" frameborder="0" allowfullscreen="allowfullscreen"></iframe> <iframe width="320" height="180" src="https://www.youtube.com/embed/4xgRG9qVT5E"></iframe></p>
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'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<p><strong>Reduced representation bisulfite sequencing (RRBS) </strong> <span>enables </span><span>genome-s</span><span>cale </span>DNA methylation<span> analysis</span> at the single nucleotide level <span>in any vertebrate species. </span><span>The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of</span> whole genome sequencing. By cutting the genome using the restriction MspI enzyme (CCGG target sites) followed by size selection, DNA is enriched to represent<span> biologically relevant target</span> CpG-rich regions including <span>promoters and </span>CpG islands.<span> Our RRBS service makes this technology widely available and provides high coverage (up to 7 million CpGs</span><span> detected </span><span>in human samples).</span></p>
<h2>Genome-scale DNA methylation analysis</h2>
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<h2>Complete end-to-end service</h2>
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<li><span><span class="ui-provider ee cfi bsv cfj cfk cfl cfm cfn cfo cfp cfq cfr cfs cft cfu cfv cfw cfx cfy cfz cga cgb cgc cgd cge cgf cgg cgh cgi cgj cgk cgl cgm cgn cgo" dir="ltr">From DNA QC to sequencing raw data</span></span></li>
<li>Leveraging our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> widely adopted</li>
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<h2></h2>
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<p><span><i class="fa fa-arrow-circle-right"></i> </span><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">See our other DNA Methylation Profiling Services</a></p>',
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<li>7 million CpGs (on average) for human samples</li>
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<p><span>Premium Methyl UDI-UMI Adapters Module - Set A includes 24 m</span>ethylated full-length adapters <span>with <strong>unique dual indexes</strong> (UDI) and<strong> unique molecular identifiers</strong> (UMI) for library multiplexing with the <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> and other BS-seq applications</span><span>. The use of </span>UDIs (unique i5 and i7 barcodes) is<span> highly recommended to mitigate errors introduced by read misassignment. The use of UMIs (9-base) is hilghy recommended for quantitave analysis or low-frequency variant detection.</span></p>
<p><span>Read more about </span><a href="https://www.diagenode.com/en/categories/bisulfite-conversion" target="_blank">Diagenode's BS-seq tools</a><span>.</span><span> </span></p>
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<p><span>UMIs incorporate a unique barcode onto each molecule within a given RRBS library. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original DNA sample (true variants) can be distinguished from errors introduced during library preparation or sequencing.</span></p>
<p><span></span></p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
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</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
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<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
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</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
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<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<h2>Epigenetics and Cancer</h2>
<p>Epigenetic processes control the normal development and maintenance of gene expression in a number of organisms. However, disruption of epigenetic mechanisms may alter gene function, potentially leading to cellular transformations that lead to tumorigenesis. Cancer is a multistep process from a succession of processes that alter the growth of normal cells. In cancer, epigenetic reprogramming occurs at multiple levels including nuclear organization and nucleosome positioning, DNA methylation, modification of histones, alteration of epigenetic regulators such as transcription factor binding, and non-coding RNA, such as microRNA.</p>
<p>Epigenetic modifications are reversible and potential treatments could be geared to alter irregular transcription factor binding, DNA methylation or histone acetylation. Accurate study with a versatile number of tools is essential for the study of epigenetics including sound sample preparation and analysis methodologies for DNA methylation such as with immunoprecipitation or bisulfite sequencing, chromatin analysis with chromatin immunoprecipitation (ChIP) combined with qPCR or sequencing, and RNA modification study. As researchers elucidate the mechanisms and specific modifications associated with cancer, it may be possible to target abnormal modifications in cells with minimal damage to normal cells, making the prospect of epigenetic therapy increasingly promising.</p>
<h3>Diagenode products for your epigenomics research in Cancer</h3>
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<center><a href="https://www.diagenode.com/en/categories/chromatin-function"><img src="https://www.diagenode.com/img/cancer/chromatin-icon.png" /></a></center>
<p class="text-left">Understand the role of chromatin in cancer regulation</p>
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<li>New to ChIP? <a href="https://www.diagenode.com/en/pages/portal/chip">Learn more</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-shearing">Chromatin shearing optimization</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-immunoprecipitation">Chromatin Immunoprecipitation</a></li>
<li>Validated <a href="https://www.diagenode.com/en/categories/antibodies">Antibodies</a></li>
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<center><a href="https://www.diagenode.com/en/categories/dna-methylation"><img src="https://www.diagenode.com/img/cancer/dna-icon.png" /></a></center>
<p class="text-left">Analyze DNA methylation and the effects on oncogenesis and tumor suppression</p>
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<li><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">DNA methylation services</a></li>
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<h3 class="text-center"><span class="darkgrey">Non-coding RNAs</span></h3>
<center><img src="https://www.diagenode.com/img/cancer/non-coding-icon.png" /></center>
<p class="text-left">Discover noncoding RNAs in cancer</p>
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<li><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">RNA-seq using D-Plex</a> </li>
<li><a href="https://www.diagenode.com/en/categories/rna-seq-category">RNA-seq services</a></li>
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<h6 style="height:60px">RRBS受託サービス (Reduced Representation Bisulfite Se...</h6>
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<li>
<div class="row">
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<span class="success label" style="">C02030040</span>
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<p><strong><input name="data[Cart][quantity]" placeholder="1" value="1" min="1" style="width:60px;display:inline" type="number" id="CartQuantity" required="required"/></strong>Premium Methyl UDI-UMI Adapters Module - Set A個カートに追加。</p>
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<h6 style="height:60px">Premium Methyl UMI-UDI Adapters - Set A</h6>
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'
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/RRBS-KIT-V2_manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><span>Premium Methyl UDI-UMI Adapters Module - Set A includes 24 m</span>ethylated full-length adapters <span>with <strong>unique dual indexes</strong> (UDI) and<strong> unique molecular identifiers</strong> (UMI) for library multiplexing with the <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> and other BS-seq applications</span><span>. The use of </span>UDIs (unique i5 and i7 barcodes) is<span> highly recommended to mitigate errors introduced by read misassignment. The use of UMIs (9-base) is hilghy recommended for quantitave analysis or low-frequency variant detection.</span></p>
<p><span>Read more about </span><a href="https://www.diagenode.com/en/categories/bisulfite-conversion" target="_blank">Diagenode's BS-seq tools</a><span>.</span><span> </span></p>
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<p><span>UMIs incorporate a unique barcode onto each molecule within a given RRBS library. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original DNA sample (true variants) can be distinguished from errors introduced during library preparation or sequencing.</span></p>
<p><span></span></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/construct_rrbs.png" /></p>
<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
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<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-96-rxns">C02030042 - Premium Methyl UMI-UDI Adapters - 96 rxns</a></li>
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<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
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<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
'label2' => 'How it works?',
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<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
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<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<p><strong>Reduced representation bisulfite sequencing (RRBS) </strong> <span>enables </span><span>genome-s</span><span>cale </span>DNA methylation<span> analysis</span> at the single nucleotide level <span>in any vertebrate species. </span><span>The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of</span> whole genome sequencing. By cutting the genome using the restriction MspI enzyme (CCGG target sites) followed by size selection, DNA is enriched to represent<span> biologically relevant target</span> CpG-rich regions including <span>promoters and </span>CpG islands.<span> Our RRBS service makes this technology widely available and provides high coverage (up to 7 million CpGs</span><span> detected </span><span>in human samples).</span></p>
<h2>Genome-scale DNA methylation analysis</h2>
<ul class="square">
<li>Robust and cost-effective solution with reliable results</li>
<li>Capability to use gDNA inputs down to 25 ng</li>
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<li>UMI technology eliminates PCR duplicates</li>
<li>High coverage - up to 7 million CpGs detected in human samples</li>
<li>Single nucleotide resolution</li>
<li>Detection of methylation patterns in CpG rich regions including promoters and CpG islands</li>
<li>Suitable for any vertebrate species</li>
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<h2>Complete end-to-end service</h2>
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<li><span><span class="ui-provider ee cfi bsv cfj cfk cfl cfm cfn cfo cfp cfq cfr cfs cft cfu cfv cfw cfx cfy cfz cga cgb cgc cgd cge cgf cgg cgh cgi cgj cgk cgl cgm cgn cgo" dir="ltr">From DNA QC to sequencing raw data</span></span></li>
<li>Leveraging our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> widely adopted</li>
<li>As little as 25 ng for any vertebrate species</li>
<li>Differentially methylated site analysis <span>using <a href="https://www.diagenode.com/en/categories/bioinformatics-service">bioinformatics tools</a></span></li>
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<h2></h2>
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<p><span><i class="fa fa-arrow-circle-right"></i> </span><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">See our other DNA Methylation Profiling Services</a></p>',
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<h4>RRBS Service includes:</h4>
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<td style="width: 264px;"><strong>QC of the genomic DNA</strong></td>
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<li>Measurement of DNA concentration </li>
<li>Assessment of DNA quality</li>
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<td style="width: 264px;"><strong>Preparation of RRBS libraries</strong></td>
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<li>MspI digestion</li>
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<li>Sample pooling</li>
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<td style="width: 264px;"><strong>Deep sequencing</strong></td>
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<li>Samples are sequenced on an Illumina platform, paired-end reads, read length 50 bp (PE50)</li>
<li>40 million raw reads (on average) per sample when pooling 10 samples/lane</li>
<li>7 million CpGs (on average) for human samples</li>
<li>7-11x CpG coverage (on average) for human samples</li>
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<h4><strong>Analysis</strong></h4>
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<td style="width: 624px;">
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<li>FASTQC quality control insights</li>
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<li>Summary statistics</li>
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<td style="width: 262px;"><strong>Differential methylation analysis<br /></strong></td>
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<li>Differentially Methylated CpGs (DMCs) analysis</li>
<li>Differentially Methylated Regions (DMRs) analysis</li>
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<p><strong>Gene ontology terms analysis</strong></p>
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<li>Get functional insights</li>
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<p><strong>Pathway analysis</strong></p>
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<td style="width: 624px;">
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<p><span>Premium Methyl UDI-UMI Adapters Module - Set A includes 24 m</span>ethylated full-length adapters <span>with <strong>unique dual indexes</strong> (UDI) and<strong> unique molecular identifiers</strong> (UMI) for library multiplexing with the <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> and other BS-seq applications</span><span>. The use of </span>UDIs (unique i5 and i7 barcodes) is<span> highly recommended to mitigate errors introduced by read misassignment. The use of UMIs (9-base) is hilghy recommended for quantitave analysis or low-frequency variant detection.</span></p>
<p><span>Read more about </span><a href="https://www.diagenode.com/en/categories/bisulfite-conversion" target="_blank">Diagenode's BS-seq tools</a><span>.</span><span> </span></p>
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<p><span>UMIs incorporate a unique barcode onto each molecule within a given RRBS library. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original DNA sample (true variants) can be distinguished from errors introduced during library preparation or sequencing.</span></p>
<p><span></span></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/construct_rrbs.png" /></p>
<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
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</li>
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<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
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<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
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<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
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</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
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<h2>Advantages</h2>
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<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
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<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<h2>Epigenetics and Cancer</h2>
<p>Epigenetic processes control the normal development and maintenance of gene expression in a number of organisms. However, disruption of epigenetic mechanisms may alter gene function, potentially leading to cellular transformations that lead to tumorigenesis. Cancer is a multistep process from a succession of processes that alter the growth of normal cells. In cancer, epigenetic reprogramming occurs at multiple levels including nuclear organization and nucleosome positioning, DNA methylation, modification of histones, alteration of epigenetic regulators such as transcription factor binding, and non-coding RNA, such as microRNA.</p>
<p>Epigenetic modifications are reversible and potential treatments could be geared to alter irregular transcription factor binding, DNA methylation or histone acetylation. Accurate study with a versatile number of tools is essential for the study of epigenetics including sound sample preparation and analysis methodologies for DNA methylation such as with immunoprecipitation or bisulfite sequencing, chromatin analysis with chromatin immunoprecipitation (ChIP) combined with qPCR or sequencing, and RNA modification study. As researchers elucidate the mechanisms and specific modifications associated with cancer, it may be possible to target abnormal modifications in cells with minimal damage to normal cells, making the prospect of epigenetic therapy increasingly promising.</p>
<h3>Diagenode products for your epigenomics research in Cancer</h3>
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<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/chromatin-function">Chromatin analysis</a></h3>
<center><a href="https://www.diagenode.com/en/categories/chromatin-function"><img src="https://www.diagenode.com/img/cancer/chromatin-icon.png" /></a></center>
<p class="text-left">Understand the role of chromatin in cancer regulation</p>
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<li>New to ChIP? <a href="https://www.diagenode.com/en/pages/portal/chip">Learn more</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-shearing">Chromatin shearing optimization</a></li>
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<li>Validated <a href="https://www.diagenode.com/en/categories/antibodies">Antibodies</a></li>
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<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/dna-methylation" style="color: #30415c;">DNA methylation</a></h3>
<center><a href="https://www.diagenode.com/en/categories/dna-methylation"><img src="https://www.diagenode.com/img/cancer/dna-icon.png" /></a></center>
<p class="text-left">Analyze DNA methylation and the effects on oncogenesis and tumor suppression</p>
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<li><a href="https://www.diagenode.com/en/categories/methylated-dna-immunoprecipitation">Methylated DNA immunoprecipitation</a></li>
<li><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">DNA methylation services</a></li>
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<center><img src="https://www.diagenode.com/img/cancer/non-coding-icon.png" /></center>
<p class="text-left">Discover noncoding RNAs in cancer</p>
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<li><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">RNA-seq using D-Plex</a> </li>
<li><a href="https://www.diagenode.com/en/categories/rna-seq-category">RNA-seq services</a></li>
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<option value="AX">Åland Islands</option>
<option value="AL">Albania</option>
<option value="DZ">Algeria</option>
<option value="AS">American Samoa</option>
<option value="AD">Andorra</option>
<option value="AO">Angola</option>
<option value="AI">Anguilla</option>
<option value="AQ">Antarctica</option>
<option value="AG">Antigua and Barbuda</option>
<option value="AR">Argentina</option>
<option value="AM">Armenia</option>
<option value="AW">Aruba</option>
<option value="AU">Australia</option>
<option value="AT">Austria</option>
<option value="AZ">Azerbaijan</option>
<option value="BS">Bahamas</option>
<option value="BH">Bahrain</option>
<option value="BD">Bangladesh</option>
<option value="BB">Barbados</option>
<option value="BY">Belarus</option>
<option value="BE">Belgium</option>
<option value="BZ">Belize</option>
<option value="BJ">Benin</option>
<option value="BM">Bermuda</option>
<option value="BT">Bhutan</option>
<option value="BO">Bolivia</option>
<option value="BQ">Bonaire, Sint Eustatius and Saba</option>
<option value="BA">Bosnia and Herzegovina</option>
<option value="BW">Botswana</option>
<option value="BV">Bouvet Island</option>
<option value="BR">Brazil</option>
<option value="IO">British Indian Ocean Territory</option>
<option value="BN">Brunei Darussalam</option>
<option value="BG">Bulgaria</option>
<option value="BF">Burkina Faso</option>
<option value="BI">Burundi</option>
<option value="KH">Cambodia</option>
<option value="CM">Cameroon</option>
<option value="CA">Canada</option>
<option value="CV">Cape Verde</option>
<option value="KY">Cayman Islands</option>
<option value="CF">Central African Republic</option>
<option value="TD">Chad</option>
<option value="CL">Chile</option>
<option value="CN">China</option>
<option value="CX">Christmas Island</option>
<option value="CC">Cocos (Keeling) Islands</option>
<option value="CO">Colombia</option>
<option value="KM">Comoros</option>
<option value="CG">Congo</option>
<option value="CD">Congo, The Democratic Republic of the</option>
<option value="CK">Cook Islands</option>
<option value="CR">Costa Rica</option>
<option value="CI">Côte d'Ivoire</option>
<option value="HR">Croatia</option>
<option value="CU">Cuba</option>
<option value="CW">Curaçao</option>
<option value="CY">Cyprus</option>
<option value="CZ">Czech Republic</option>
<option value="DK">Denmark</option>
<option value="DJ">Djibouti</option>
<option value="DM">Dominica</option>
<option value="DO">Dominican Republic</option>
<option value="EC">Ecuador</option>
<option value="EG">Egypt</option>
<option value="SV">El Salvador</option>
<option value="GQ">Equatorial Guinea</option>
<option value="ER">Eritrea</option>
<option value="EE">Estonia</option>
<option value="ET">Ethiopia</option>
<option value="FK">Falkland Islands (Malvinas)</option>
<option value="FO">Faroe Islands</option>
<option value="FJ">Fiji</option>
<option value="FI">Finland</option>
<option value="FR">France</option>
<option value="GF">French Guiana</option>
<option value="PF">French Polynesia</option>
<option value="TF">French Southern Territories</option>
<option value="GA">Gabon</option>
<option value="GM">Gambia</option>
<option value="GE">Georgia</option>
<option value="DE">Germany</option>
<option value="GH">Ghana</option>
<option value="GI">Gibraltar</option>
<option value="GR">Greece</option>
<option value="GL">Greenland</option>
<option value="GD">Grenada</option>
<option value="GP">Guadeloupe</option>
<option value="GU">Guam</option>
<option value="GT">Guatemala</option>
<option value="GG">Guernsey</option>
<option value="GN">Guinea</option>
<option value="GW">Guinea-Bissau</option>
<option value="GY">Guyana</option>
<option value="HT">Haiti</option>
<option value="HM">Heard Island and McDonald Islands</option>
<option value="VA">Holy See (Vatican City State)</option>
<option value="HN">Honduras</option>
<option value="HK">Hong Kong</option>
<option value="HU">Hungary</option>
<option value="IS">Iceland</option>
<option value="IN">India</option>
<option value="ID">Indonesia</option>
<option value="IR">Iran, Islamic Republic of</option>
<option value="IQ">Iraq</option>
<option value="IE">Ireland</option>
<option value="IM">Isle of Man</option>
<option value="IL">Israel</option>
<option value="IT">Italy</option>
<option value="JM">Jamaica</option>
<option value="JP">Japan</option>
<option value="JE">Jersey</option>
<option value="JO">Jordan</option>
<option value="KZ">Kazakhstan</option>
<option value="KE">Kenya</option>
<option value="KI">Kiribati</option>
<option value="KP">Korea, Democratic People's Republic of</option>
<option value="KR">Korea, Republic of</option>
<option value="KW">Kuwait</option>
<option value="KG">Kyrgyzstan</option>
<option value="LA">Lao People's Democratic Republic</option>
<option value="LV">Latvia</option>
<option value="LB">Lebanon</option>
<option value="LS">Lesotho</option>
<option value="LR">Liberia</option>
<option value="LY">Libya</option>
<option value="LI">Liechtenstein</option>
<option value="LT">Lithuania</option>
<option value="LU">Luxembourg</option>
<option value="MO">Macao</option>
<option value="MK">Macedonia, Republic of</option>
<option value="MG">Madagascar</option>
<option value="MW">Malawi</option>
<option value="MY">Malaysia</option>
<option value="MV">Maldives</option>
<option value="ML">Mali</option>
<option value="MT">Malta</option>
<option value="MH">Marshall Islands</option>
<option value="MQ">Martinique</option>
<option value="MR">Mauritania</option>
<option value="MU">Mauritius</option>
<option value="YT">Mayotte</option>
<option value="MX">Mexico</option>
<option value="FM">Micronesia, Federated States of</option>
<option value="MD">Moldova</option>
<option value="MC">Monaco</option>
<option value="MN">Mongolia</option>
<option value="ME">Montenegro</option>
<option value="MS">Montserrat</option>
<option value="MA">Morocco</option>
<option value="MZ">Mozambique</option>
<option value="MM">Myanmar</option>
<option value="NA">Namibia</option>
<option value="NR">Nauru</option>
<option value="NP">Nepal</option>
<option value="NL">Netherlands</option>
<option value="NC">New Caledonia</option>
<option value="NZ">New Zealand</option>
<option value="NI">Nicaragua</option>
<option value="NE">Niger</option>
<option value="NG">Nigeria</option>
<option value="NU">Niue</option>
<option value="NF">Norfolk Island</option>
<option value="MP">Northern Mariana Islands</option>
<option value="NO">Norway</option>
<option value="OM">Oman</option>
<option value="PK">Pakistan</option>
<option value="PW">Palau</option>
<option value="PS">Palestine, State of</option>
<option value="PA">Panama</option>
<option value="PG">Papua New Guinea</option>
<option value="PY">Paraguay</option>
<option value="PE">Peru</option>
<option value="PH">Philippines</option>
<option value="PN">Pitcairn</option>
<option value="PL">Poland</option>
<option value="PT">Portugal</option>
<option value="PR">Puerto Rico</option>
<option value="QA">Qatar</option>
<option value="RE">Réunion</option>
<option value="RO">Romania</option>
<option value="RU">Russian Federation</option>
<option value="RW">Rwanda</option>
<option value="BL">Saint Barthélemy</option>
<option value="SH">Saint Helena, Ascension and Tristan da Cunha</option>
<option value="KN">Saint Kitts and Nevis</option>
<option value="LC">Saint Lucia</option>
<option value="MF">Saint Martin (French part)</option>
<option value="PM">Saint Pierre and Miquelon</option>
<option value="VC">Saint Vincent and the Grenadines</option>
<option value="WS">Samoa</option>
<option value="SM">San Marino</option>
<option value="ST">Sao Tome and Principe</option>
<option value="SA">Saudi Arabia</option>
<option value="SN">Senegal</option>
<option value="RS">Serbia</option>
<option value="SC">Seychelles</option>
<option value="SL">Sierra Leone</option>
<option value="SG">Singapore</option>
<option value="SX">Sint Maarten (Dutch part)</option>
<option value="SK">Slovakia</option>
<option value="SI">Slovenia</option>
<option value="SB">Solomon Islands</option>
<option value="SO">Somalia</option>
<option value="ZA">South Africa</option>
<option value="GS">South Georgia and the South Sandwich Islands</option>
<option value="ES">Spain</option>
<option value="LK">Sri Lanka</option>
<option value="SD">Sudan</option>
<option value="SR">Suriname</option>
<option value="SS">South Sudan</option>
<option value="SJ">Svalbard and Jan Mayen</option>
<option value="SZ">Swaziland</option>
<option value="SE">Sweden</option>
<option value="CH">Switzerland</option>
<option value="SY">Syrian Arab Republic</option>
<option value="TW">Taiwan</option>
<option value="TJ">Tajikistan</option>
<option value="TZ">Tanzania</option>
<option value="TH">Thailand</option>
<option value="TL">Timor-Leste</option>
<option value="TG">Togo</option>
<option value="TK">Tokelau</option>
<option value="TO">Tonga</option>
<option value="TT">Trinidad and Tobago</option>
<option value="TN">Tunisia</option>
<option value="TR">Turkey</option>
<option value="TM">Turkmenistan</option>
<option value="TC">Turks and Caicos Islands</option>
<option value="TV">Tuvalu</option>
<option value="UG">Uganda</option>
<option value="UA">Ukraine</option>
<option value="AE">United Arab Emirates</option>
<option value="GB">United Kingdom</option>
<option value="US" selected="selected">United States</option>
<option value="UM">United States Minor Outlying Islands</option>
<option value="UY">Uruguay</option>
<option value="UZ">Uzbekistan</option>
<option value="VU">Vanuatu</option>
<option value="VE">Venezuela</option>
<option value="VN">Viet Nam</option>
<option value="VG">Virgin Islands, British</option>
<option value="VI">Virgin Islands, U.S.</option>
<option value="WF">Wallis and Futuna</option>
<option value="EH">Western Sahara</option>
<option value="YE">Yemen</option>
<option value="ZM">Zambia</option>
<option value="ZW">Zimbabwe</option>
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$('#country_selector_quote-2836').selectize();
</script><br />
</div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">State</span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][state]" id="state-2836" maxlength="3" type="text"/><br />
</div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Email <sup style="font-size:16px;color:red;">*</sup></span>
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<div class="small-9 large-10 columns">
<input name="data[Quote][email]" placeholder="email@address.com" maxlength="255" type="email" id="QuoteEmail" required="required"/> </div>
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<div class="row collapse" id="email_v">
<div class="small-3 large-2 columns">
<span class="prefix">Email verification<sup style="font-size:16px;color:red;">*</sup></span>
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<div class="small-9 large-10 columns">
<input name="data[Quote][email_v]" autocomplete="nope" type="text" id="QuoteEmailV"/> </div>
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<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Project</span>
</div>
<div class="small-9 large-10 columns">
<textarea name="data[Quote][comment]" placeholder="Describe your project" cols="30" rows="6" id="QuoteComment"></textarea> </div>
</div>
<!------------SERVICES PARTICULAR FORM START---------------->
<!------------DATA TO POPULATE REGARDING SPECIFIC SERVICES----->
<div class="row collapse">
<div class="small-3 large-2 columns">
</div>
<div class="small-9 large-10 columns">
<div class="recaptcha"><div id="recaptcha673efae88ac38"></div></div> </div>
</div>
<br />
<div class="row collapse">
<div class="small-3 large-2 columns">
</div>
<div class="small-9 large-10 columns">
<button id="submit_btn-2836" class="alert button expand" form="Quote-2836" type="submit">Contact me</button> </div>
</div>
</form><script>
var pardotFormHandlerURL = 'https://go.diagenode.com/l/928883/2022-10-10/36b1c';
function postToPardot(formAction, id) {
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$('#Quote-' + id).submit();
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$('#pardot-form-handler').attr('src', pardotFormHandlerURL + '?' + $('#Quote-' + id).serialize());
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$(document).ready(function() {
$('body').append('<iframe id="pardot-form-handler" height="0" width="0" style="position:absolute; left:-100000px; top:-100000px" src="javascript:false;"></iframe>');
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<a class="close-reveal-modal" aria-label="Close">×</a></div><!-- END: QUOTE MODAL --><a href="#" id="rrbs-service" data-reveal-id="quoteModal-2836" class="quote_btn" style="color:#B21329"><i class="fa fa-info-circle"></i></a>
</div>
</div>
<div class="small-12 columns" >
<h6 style="height:60px">RRBS受託サービス (Reduced Representation Bisulfite Se...</h6>
</div>
</div>
</li>
<li>
<div class="row">
<div class="small-12 columns">
<a href="/jp/p/premium-methyl-UDI-UMI-adapters-set-A-x24"><img src="/img/grey-logo.jpg" alt="default alt" class="th"/></a> </div>
<div class="small-12 columns">
<div class="small-6 columns" style="padding-left:0px;padding-right:0px;margin-top:-6px;margin-left:-1px">
<span class="success label" style="">C02030040</span>
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<div class="small-6 columns text-right" style="padding-left:0px;padding-right:0px;margin-top:-6px">
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
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<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-96-rxns">C02030042 - Premium Methyl UMI-UDI Adapters - 96 rxns</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
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<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
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<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
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<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
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<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<p><strong>Reduced representation bisulfite sequencing (RRBS) </strong> <span>enables </span><span>genome-s</span><span>cale </span>DNA methylation<span> analysis</span> at the single nucleotide level <span>in any vertebrate species. </span><span>The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of</span> whole genome sequencing. By cutting the genome using the restriction MspI enzyme (CCGG target sites) followed by size selection, DNA is enriched to represent<span> biologically relevant target</span> CpG-rich regions including <span>promoters and </span>CpG islands.<span> Our RRBS service makes this technology widely available and provides high coverage (up to 7 million CpGs</span><span> detected </span><span>in human samples).</span></p>
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<li>Capability to use gDNA inputs down to 25 ng</li>
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<li>High coverage - up to 7 million CpGs detected in human samples</li>
<li>Single nucleotide resolution</li>
<li>Detection of methylation patterns in CpG rich regions including promoters and CpG islands</li>
<li>Suitable for any vertebrate species</li>
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<h2>Complete end-to-end service</h2>
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<li><span><span class="ui-provider ee cfi bsv cfj cfk cfl cfm cfn cfo cfp cfq cfr cfs cft cfu cfv cfw cfx cfy cfz cga cgb cgc cgd cge cgf cgg cgh cgi cgj cgk cgl cgm cgn cgo" dir="ltr">From DNA QC to sequencing raw data</span></span></li>
<li>Leveraging our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> widely adopted</li>
<li>As little as 25 ng for any vertebrate species</li>
<li>Differentially methylated site analysis <span>using <a href="https://www.diagenode.com/en/categories/bioinformatics-service">bioinformatics tools</a></span></li>
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<h2></h2>
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<p><span><i class="fa fa-arrow-circle-right"></i> </span><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">See our other DNA Methylation Profiling Services</a></p>',
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<li>Assessment of DNA quality</li>
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<li>40 million raw reads (on average) per sample when pooling 10 samples/lane</li>
<li>7 million CpGs (on average) for human samples</li>
<li>7-11x CpG coverage (on average) for human samples</li>
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<h4><strong>Analysis</strong></h4>
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<h4><strong>Features</strong></h4>
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<td style="width: 624px;">
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<li>Get functional insights</li>
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<p><strong>Pathway analysis</strong></p>
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<p><span>Premium Methyl UDI-UMI Adapters Module - Set A includes 24 m</span>ethylated full-length adapters <span>with <strong>unique dual indexes</strong> (UDI) and<strong> unique molecular identifiers</strong> (UMI) for library multiplexing with the <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> and other BS-seq applications</span><span>. The use of </span>UDIs (unique i5 and i7 barcodes) is<span> highly recommended to mitigate errors introduced by read misassignment. The use of UMIs (9-base) is hilghy recommended for quantitave analysis or low-frequency variant detection.</span></p>
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<p><span></span></p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
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<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
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<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<p><img src="https://www.diagenode.com/img/areas/cancer.jpg" /></p>
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<h2>Epigenetics and Cancer</h2>
<p>Epigenetic processes control the normal development and maintenance of gene expression in a number of organisms. However, disruption of epigenetic mechanisms may alter gene function, potentially leading to cellular transformations that lead to tumorigenesis. Cancer is a multistep process from a succession of processes that alter the growth of normal cells. In cancer, epigenetic reprogramming occurs at multiple levels including nuclear organization and nucleosome positioning, DNA methylation, modification of histones, alteration of epigenetic regulators such as transcription factor binding, and non-coding RNA, such as microRNA.</p>
<p>Epigenetic modifications are reversible and potential treatments could be geared to alter irregular transcription factor binding, DNA methylation or histone acetylation. Accurate study with a versatile number of tools is essential for the study of epigenetics including sound sample preparation and analysis methodologies for DNA methylation such as with immunoprecipitation or bisulfite sequencing, chromatin analysis with chromatin immunoprecipitation (ChIP) combined with qPCR or sequencing, and RNA modification study. As researchers elucidate the mechanisms and specific modifications associated with cancer, it may be possible to target abnormal modifications in cells with minimal damage to normal cells, making the prospect of epigenetic therapy increasingly promising.</p>
<h3>Diagenode products for your epigenomics research in Cancer</h3>
<div class="row">
<div class="small-12 medium-4 large-4 columns text-left">
<div class="panel" style="border-color: #099f92; height: 275px;">
<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/chromatin-function">Chromatin analysis</a></h3>
<center><a href="https://www.diagenode.com/en/categories/chromatin-function"><img src="https://www.diagenode.com/img/cancer/chromatin-icon.png" /></a></center>
<p class="text-left">Understand the role of chromatin in cancer regulation</p>
</div>
<ul>
<li>New to ChIP? <a href="https://www.diagenode.com/en/pages/portal/chip">Learn more</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-shearing">Chromatin shearing optimization</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-immunoprecipitation">Chromatin Immunoprecipitation</a></li>
<li>Validated <a href="https://www.diagenode.com/en/categories/antibodies">Antibodies</a></li>
<li><a href="https://www.diagenode.com/en/categories/library-preparation">Library preparation</a></li>
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<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/dna-methylation" style="color: #30415c;">DNA methylation</a></h3>
<center><a href="https://www.diagenode.com/en/categories/dna-methylation"><img src="https://www.diagenode.com/img/cancer/dna-icon.png" /></a></center>
<p class="text-left">Analyze DNA methylation and the effects on oncogenesis and tumor suppression</p>
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<li><a href="https://www.diagenode.com/en/categories/bisulfite-conversion">Bisulfite sequencing</a></li>
<li><a href="https://www.diagenode.com/en/categories/methylated-dna-immunoprecipitation">Methylated DNA immunoprecipitation</a></li>
<li><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">DNA methylation services</a></li>
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<h3 class="text-center"><span class="darkgrey">Non-coding RNAs</span></h3>
<center><img src="https://www.diagenode.com/img/cancer/non-coding-icon.png" /></center>
<p class="text-left">Discover noncoding RNAs in cancer</p>
</div>
<ul>
<li><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">RNA-seq using D-Plex</a> </li>
<li><a href="https://www.diagenode.com/en/categories/rna-seq-category">RNA-seq services</a></li>
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<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="WGBS" id="QuoteEpigenomicsServiceWGBS" /><label for="QuoteEpigenomicsServiceWGBS">WGBS</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="MeDIP-seq" id="QuoteEpigenomicsServiceMeDIPSeq" /><label for="QuoteEpigenomicsServiceMeDIPSeq">MeDIP-seq</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Targeted DNA methylation analysis" id="QuoteEpigenomicsServiceTargetedDNAMethylationAnalysis" /><label for="QuoteEpigenomicsServiceTargetedDNAMethylationAnalysis">Targeted DNA methylation analysis</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Infinium MethylationEPIC Array v2" id="QuoteEpigenomicsServiceInfiniumMethylationEPICArrayV2" /><label for="QuoteEpigenomicsServiceInfiniumMethylationEPICArrayV2">Infinium MethylationEPIC Array v2</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Infinium Mouse Methylation Array" id="QuoteEpigenomicsServiceInfiniumMouseMethylationArray" /><label for="QuoteEpigenomicsServiceInfiniumMouseMethylationArray">Infinium Mouse Methylation Array</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="RNA-seq" id="QuoteEpigenomicsServiceRNASeq" /><label for="QuoteEpigenomicsServiceRNASeq">RNA-seq</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Bioinformatics" id="QuoteEpigenomicsServiceBioinformatics" /><label for="QuoteEpigenomicsServiceBioinformatics">Bioinformatics</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Data mining" id="QuoteEpigenomicsServiceDataMining" /><label for="QuoteEpigenomicsServiceDataMining">Data mining</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Human Methylome" id="QuoteEpigenomicsServiceHumanMethylome" /><label for="QuoteEpigenomicsServiceHumanMethylome">Human Methylome</label></div>
</div>
<div class="row collapse">
<div class="small-12 medium-12 large-3 columns">
<span class="prefix">Sample species</span>
</div>
<div class="small-12 medium-12 large-9 columns">
<input name="data[Quote][sample_species]" maxlength="510" type="text" id="QuoteSampleSpecies"/> </div>
</div>
<div class="row collapse">
<div class="small-12 medium-12 large-6 columns">
<span class="prefix">Total number of samples (including replicates)</span>
</div>
<div class="small-12 medium-12 large-6 columns">
<input name="data[Quote][number_samples]" maxlength="255" type="text" id="QuoteNumberSamples"/> </div>
</div>
<div class="row collapse">
<h2>Contact Information</h2>
<div class="small-3 large-2 columns">
<span class="prefix">First name <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][first_name]" placeholder="john" maxlength="255" type="text" id="QuoteFirstName" required="required"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Last name <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][last_name]" placeholder="doe" maxlength="255" type="text" id="QuoteLastName" required="required"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Company <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][company]" placeholder="Organisation / Institute" maxlength="255" type="text" id="QuoteCompany" required="required"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Phone number</span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][phone_number]" placeholder="+1 862 209-4680" maxlength="255" type="text" id="QuotePhoneNumber"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">City</span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][city]" placeholder="Denville" maxlength="255" type="text" id="QuoteCity"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Country <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<select name="data[Quote][country]" required="required" class="triggers" id="country_selector_quote-2836">
<option value="">-- select a country --</option>
<option value="AF">Afghanistan</option>
<option value="AX">Åland Islands</option>
<option value="AL">Albania</option>
<option value="DZ">Algeria</option>
<option value="AS">American Samoa</option>
<option value="AD">Andorra</option>
<option value="AO">Angola</option>
<option value="AI">Anguilla</option>
<option value="AQ">Antarctica</option>
<option value="AG">Antigua and Barbuda</option>
<option value="AR">Argentina</option>
<option value="AM">Armenia</option>
<option value="AW">Aruba</option>
<option value="AU">Australia</option>
<option value="AT">Austria</option>
<option value="AZ">Azerbaijan</option>
<option value="BS">Bahamas</option>
<option value="BH">Bahrain</option>
<option value="BD">Bangladesh</option>
<option value="BB">Barbados</option>
<option value="BY">Belarus</option>
<option value="BE">Belgium</option>
<option value="BZ">Belize</option>
<option value="BJ">Benin</option>
<option value="BM">Bermuda</option>
<option value="BT">Bhutan</option>
<option value="BO">Bolivia</option>
<option value="BQ">Bonaire, Sint Eustatius and Saba</option>
<option value="BA">Bosnia and Herzegovina</option>
<option value="BW">Botswana</option>
<option value="BV">Bouvet Island</option>
<option value="BR">Brazil</option>
<option value="IO">British Indian Ocean Territory</option>
<option value="BN">Brunei Darussalam</option>
<option value="BG">Bulgaria</option>
<option value="BF">Burkina Faso</option>
<option value="BI">Burundi</option>
<option value="KH">Cambodia</option>
<option value="CM">Cameroon</option>
<option value="CA">Canada</option>
<option value="CV">Cape Verde</option>
<option value="KY">Cayman Islands</option>
<option value="CF">Central African Republic</option>
<option value="TD">Chad</option>
<option value="CL">Chile</option>
<option value="CN">China</option>
<option value="CX">Christmas Island</option>
<option value="CC">Cocos (Keeling) Islands</option>
<option value="CO">Colombia</option>
<option value="KM">Comoros</option>
<option value="CG">Congo</option>
<option value="CD">Congo, The Democratic Republic of the</option>
<option value="CK">Cook Islands</option>
<option value="CR">Costa Rica</option>
<option value="CI">Côte d'Ivoire</option>
<option value="HR">Croatia</option>
<option value="CU">Cuba</option>
<option value="CW">Curaçao</option>
<option value="CY">Cyprus</option>
<option value="CZ">Czech Republic</option>
<option value="DK">Denmark</option>
<option value="DJ">Djibouti</option>
<option value="DM">Dominica</option>
<option value="DO">Dominican Republic</option>
<option value="EC">Ecuador</option>
<option value="EG">Egypt</option>
<option value="SV">El Salvador</option>
<option value="GQ">Equatorial Guinea</option>
<option value="ER">Eritrea</option>
<option value="EE">Estonia</option>
<option value="ET">Ethiopia</option>
<option value="FK">Falkland Islands (Malvinas)</option>
<option value="FO">Faroe Islands</option>
<option value="FJ">Fiji</option>
<option value="FI">Finland</option>
<option value="FR">France</option>
<option value="GF">French Guiana</option>
<option value="PF">French Polynesia</option>
<option value="TF">French Southern Territories</option>
<option value="GA">Gabon</option>
<option value="GM">Gambia</option>
<option value="GE">Georgia</option>
<option value="DE">Germany</option>
<option value="GH">Ghana</option>
<option value="GI">Gibraltar</option>
<option value="GR">Greece</option>
<option value="GL">Greenland</option>
<option value="GD">Grenada</option>
<option value="GP">Guadeloupe</option>
<option value="GU">Guam</option>
<option value="GT">Guatemala</option>
<option value="GG">Guernsey</option>
<option value="GN">Guinea</option>
<option value="GW">Guinea-Bissau</option>
<option value="GY">Guyana</option>
<option value="HT">Haiti</option>
<option value="HM">Heard Island and McDonald Islands</option>
<option value="VA">Holy See (Vatican City State)</option>
<option value="HN">Honduras</option>
<option value="HK">Hong Kong</option>
<option value="HU">Hungary</option>
<option value="IS">Iceland</option>
<option value="IN">India</option>
<option value="ID">Indonesia</option>
<option value="IR">Iran, Islamic Republic of</option>
<option value="IQ">Iraq</option>
<option value="IE">Ireland</option>
<option value="IM">Isle of Man</option>
<option value="IL">Israel</option>
<option value="IT">Italy</option>
<option value="JM">Jamaica</option>
<option value="JP">Japan</option>
<option value="JE">Jersey</option>
<option value="JO">Jordan</option>
<option value="KZ">Kazakhstan</option>
<option value="KE">Kenya</option>
<option value="KI">Kiribati</option>
<option value="KP">Korea, Democratic People's Republic of</option>
<option value="KR">Korea, Republic of</option>
<option value="KW">Kuwait</option>
<option value="KG">Kyrgyzstan</option>
<option value="LA">Lao People's Democratic Republic</option>
<option value="LV">Latvia</option>
<option value="LB">Lebanon</option>
<option value="LS">Lesotho</option>
<option value="LR">Liberia</option>
<option value="LY">Libya</option>
<option value="LI">Liechtenstein</option>
<option value="LT">Lithuania</option>
<option value="LU">Luxembourg</option>
<option value="MO">Macao</option>
<option value="MK">Macedonia, Republic of</option>
<option value="MG">Madagascar</option>
<option value="MW">Malawi</option>
<option value="MY">Malaysia</option>
<option value="MV">Maldives</option>
<option value="ML">Mali</option>
<option value="MT">Malta</option>
<option value="MH">Marshall Islands</option>
<option value="MQ">Martinique</option>
<option value="MR">Mauritania</option>
<option value="MU">Mauritius</option>
<option value="YT">Mayotte</option>
<option value="MX">Mexico</option>
<option value="FM">Micronesia, Federated States of</option>
<option value="MD">Moldova</option>
<option value="MC">Monaco</option>
<option value="MN">Mongolia</option>
<option value="ME">Montenegro</option>
<option value="MS">Montserrat</option>
<option value="MA">Morocco</option>
<option value="MZ">Mozambique</option>
<option value="MM">Myanmar</option>
<option value="NA">Namibia</option>
<option value="NR">Nauru</option>
<option value="NP">Nepal</option>
<option value="NL">Netherlands</option>
<option value="NC">New Caledonia</option>
<option value="NZ">New Zealand</option>
<option value="NI">Nicaragua</option>
<option value="NE">Niger</option>
<option value="NG">Nigeria</option>
<option value="NU">Niue</option>
<option value="NF">Norfolk Island</option>
<option value="MP">Northern Mariana Islands</option>
<option value="NO">Norway</option>
<option value="OM">Oman</option>
<option value="PK">Pakistan</option>
<option value="PW">Palau</option>
<option value="PS">Palestine, State of</option>
<option value="PA">Panama</option>
<option value="PG">Papua New Guinea</option>
<option value="PY">Paraguay</option>
<option value="PE">Peru</option>
<option value="PH">Philippines</option>
<option value="PN">Pitcairn</option>
<option value="PL">Poland</option>
<option value="PT">Portugal</option>
<option value="PR">Puerto Rico</option>
<option value="QA">Qatar</option>
<option value="RE">Réunion</option>
<option value="RO">Romania</option>
<option value="RU">Russian Federation</option>
<option value="RW">Rwanda</option>
<option value="BL">Saint Barthélemy</option>
<option value="SH">Saint Helena, Ascension and Tristan da Cunha</option>
<option value="KN">Saint Kitts and Nevis</option>
<option value="LC">Saint Lucia</option>
<option value="MF">Saint Martin (French part)</option>
<option value="PM">Saint Pierre and Miquelon</option>
<option value="VC">Saint Vincent and the Grenadines</option>
<option value="WS">Samoa</option>
<option value="SM">San Marino</option>
<option value="ST">Sao Tome and Principe</option>
<option value="SA">Saudi Arabia</option>
<option value="SN">Senegal</option>
<option value="RS">Serbia</option>
<option value="SC">Seychelles</option>
<option value="SL">Sierra Leone</option>
<option value="SG">Singapore</option>
<option value="SX">Sint Maarten (Dutch part)</option>
<option value="SK">Slovakia</option>
<option value="SI">Slovenia</option>
<option value="SB">Solomon Islands</option>
<option value="SO">Somalia</option>
<option value="ZA">South Africa</option>
<option value="GS">South Georgia and the South Sandwich Islands</option>
<option value="ES">Spain</option>
<option value="LK">Sri Lanka</option>
<option value="SD">Sudan</option>
<option value="SR">Suriname</option>
<option value="SS">South Sudan</option>
<option value="SJ">Svalbard and Jan Mayen</option>
<option value="SZ">Swaziland</option>
<option value="SE">Sweden</option>
<option value="CH">Switzerland</option>
<option value="SY">Syrian Arab Republic</option>
<option value="TW">Taiwan</option>
<option value="TJ">Tajikistan</option>
<option value="TZ">Tanzania</option>
<option value="TH">Thailand</option>
<option value="TL">Timor-Leste</option>
<option value="TG">Togo</option>
<option value="TK">Tokelau</option>
<option value="TO">Tonga</option>
<option value="TT">Trinidad and Tobago</option>
<option value="TN">Tunisia</option>
<option value="TR">Turkey</option>
<option value="TM">Turkmenistan</option>
<option value="TC">Turks and Caicos Islands</option>
<option value="TV">Tuvalu</option>
<option value="UG">Uganda</option>
<option value="UA">Ukraine</option>
<option value="AE">United Arab Emirates</option>
<option value="GB">United Kingdom</option>
<option value="US" selected="selected">United States</option>
<option value="UM">United States Minor Outlying Islands</option>
<option value="UY">Uruguay</option>
<option value="UZ">Uzbekistan</option>
<option value="VU">Vanuatu</option>
<option value="VE">Venezuela</option>
<option value="VN">Viet Nam</option>
<option value="VG">Virgin Islands, British</option>
<option value="VI">Virgin Islands, U.S.</option>
<option value="WF">Wallis and Futuna</option>
<option value="EH">Western Sahara</option>
<option value="YE">Yemen</option>
<option value="ZM">Zambia</option>
<option value="ZW">Zimbabwe</option>
</select><script>
$('#country_selector_quote-2836').selectize();
</script><br />
</div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">State</span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][state]" id="state-2836" maxlength="3" type="text"/><br />
</div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Email <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][email]" placeholder="email@address.com" maxlength="255" type="email" id="QuoteEmail" required="required"/> </div>
</div>
<div class="row collapse" id="email_v">
<div class="small-3 large-2 columns">
<span class="prefix">Email verification<sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][email_v]" autocomplete="nope" type="text" id="QuoteEmailV"/> </div>
</div>
<div class="row collapse">
<div class="small-3 large-2 columns">
<span class="prefix">Project</span>
</div>
<div class="small-9 large-10 columns">
<textarea name="data[Quote][comment]" placeholder="Describe your project" cols="30" rows="6" id="QuoteComment"></textarea> </div>
</div>
<!------------SERVICES PARTICULAR FORM START---------------->
<!------------DATA TO POPULATE REGARDING SPECIFIC SERVICES----->
<div class="row collapse">
<div class="small-3 large-2 columns">
</div>
<div class="small-9 large-10 columns">
<div class="recaptcha"><div id="recaptcha673efae88ac38"></div></div> </div>
</div>
<br />
<div class="row collapse">
<div class="small-3 large-2 columns">
</div>
<div class="small-9 large-10 columns">
<button id="submit_btn-2836" class="alert button expand" form="Quote-2836" type="submit">Contact me</button> </div>
</div>
</form><script>
var pardotFormHandlerURL = 'https://go.diagenode.com/l/928883/2022-10-10/36b1c';
function postToPardot(formAction, id) {
$('#pardot-form-handler').load(function(){
$('#Quote-' + id).attr('action', formAction);
$('#Quote-' + id).submit();
});
$('#pardot-form-handler').attr('src', pardotFormHandlerURL + '?' + $('#Quote-' + id).serialize());
}
$(document).ready(function() {
$('body').append('<iframe id="pardot-form-handler" height="0" width="0" style="position:absolute; left:-100000px; top:-100000px" src="javascript:false;"></iframe>');
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//disable the submit button
$("#Quote-2836 #submit_btn-2836").attr("disabled", true);
$("#Quote-2836 #submit_btn-2836").html("Processing...");
//submit the form
$("#Quote-2836").submit();
}
})
</script>
<script>
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-96-rxns">C02030042 - Premium Methyl UMI-UDI Adapters - 96 rxns</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
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<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<p><strong>Reduced representation bisulfite sequencing (RRBS) </strong> <span>enables </span><span>genome-s</span><span>cale </span>DNA methylation<span> analysis</span> at the single nucleotide level <span>in any vertebrate species. </span><span>The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of</span> whole genome sequencing. By cutting the genome using the restriction MspI enzyme (CCGG target sites) followed by size selection, DNA is enriched to represent<span> biologically relevant target</span> CpG-rich regions including <span>promoters and </span>CpG islands.<span> Our RRBS service makes this technology widely available and provides high coverage (up to 7 million CpGs</span><span> detected </span><span>in human samples).</span></p>
<h2>Genome-scale DNA methylation analysis</h2>
<ul class="square">
<li>Robust and cost-effective solution with reliable results</li>
<li>Capability to use gDNA inputs down to 25 ng</li>
<li>Use of UDIs enhance sequencing results</li>
<li>UMI technology eliminates PCR duplicates</li>
<li>High coverage - up to 7 million CpGs detected in human samples</li>
<li>Single nucleotide resolution</li>
<li>Detection of methylation patterns in CpG rich regions including promoters and CpG islands</li>
<li>Suitable for any vertebrate species</li>
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<h2>Complete end-to-end service</h2>
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<li><span><span class="ui-provider ee cfi bsv cfj cfk cfl cfm cfn cfo cfp cfq cfr cfs cft cfu cfv cfw cfx cfy cfz cga cgb cgc cgd cge cgf cgg cgh cgi cgj cgk cgl cgm cgn cgo" dir="ltr">From DNA QC to sequencing raw data</span></span></li>
<li>Leveraging our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> widely adopted</li>
<li>As little as 25 ng for any vertebrate species</li>
<li>Differentially methylated site analysis <span>using <a href="https://www.diagenode.com/en/categories/bioinformatics-service">bioinformatics tools</a></span></li>
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<div class="extra-spaced">
<h2></h2>
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<p><span><i class="fa fa-arrow-circle-right"></i> </span><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">See our other DNA Methylation Profiling Services</a></p>',
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<h4>RRBS Service includes:</h4>
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<td style="width: 264px;"><strong>QC of the genomic DNA</strong></td>
<td style="width: 636px;">
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<li>Measurement of DNA concentration </li>
<li>Assessment of DNA quality</li>
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<td style="width: 264px;"><strong>Preparation of RRBS libraries</strong></td>
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<li>MspI digestion</li>
<li>Library preparation (ends preparation, adaptor ligation)</li>
<li>Size selection</li>
<li>Sample pooling</li>
<li>Bisulfite conversion</li>
<li>Library amplification and clean-up</li>
<li>QC of the RRBS library pool (DNA concentration, analysis of the pool profile)</li>
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<td style="width: 264px;"><strong>Deep sequencing</strong></td>
<td style="width: 636px;">
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<li>Samples are sequenced on an Illumina platform, paired-end reads, read length 50 bp (PE50)</li>
<li>40 million raw reads (on average) per sample when pooling 10 samples/lane</li>
<li>7 million CpGs (on average) for human samples</li>
<li>7-11x CpG coverage (on average) for human samples</li>
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<h4><strong>Analysis</strong></h4>
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<h4><strong>Features</strong></h4>
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<li>Methylation calling and extraction</li>
<li>Summary statistics</li>
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<td style="width: 262px;"><strong>Differential methylation analysis<br /></strong></td>
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<li>Differentially Methylated CpGs (DMCs) analysis</li>
<li>Differentially Methylated Regions (DMRs) analysis</li>
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<li>Get functional insights</li>
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<p><span>Premium Methyl UDI-UMI Adapters Module - Set A includes 24 m</span>ethylated full-length adapters <span>with <strong>unique dual indexes</strong> (UDI) and<strong> unique molecular identifiers</strong> (UMI) for library multiplexing with the <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2</a> and other BS-seq applications</span><span>. The use of </span>UDIs (unique i5 and i7 barcodes) is<span> highly recommended to mitigate errors introduced by read misassignment. The use of UMIs (9-base) is hilghy recommended for quantitave analysis or low-frequency variant detection.</span></p>
<p><span>Read more about </span><a href="https://www.diagenode.com/en/categories/bisulfite-conversion" target="_blank">Diagenode's BS-seq tools</a><span>.</span><span> </span></p>
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<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
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<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
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<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
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</li>
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<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
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<p><span style="font-weight: 400;"> </span></p>
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<th></th>
<th>Description</th>
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<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
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<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
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</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
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<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
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<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
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<h2>Epigenetics and Cancer</h2>
<p>Epigenetic processes control the normal development and maintenance of gene expression in a number of organisms. However, disruption of epigenetic mechanisms may alter gene function, potentially leading to cellular transformations that lead to tumorigenesis. Cancer is a multistep process from a succession of processes that alter the growth of normal cells. In cancer, epigenetic reprogramming occurs at multiple levels including nuclear organization and nucleosome positioning, DNA methylation, modification of histones, alteration of epigenetic regulators such as transcription factor binding, and non-coding RNA, such as microRNA.</p>
<p>Epigenetic modifications are reversible and potential treatments could be geared to alter irregular transcription factor binding, DNA methylation or histone acetylation. Accurate study with a versatile number of tools is essential for the study of epigenetics including sound sample preparation and analysis methodologies for DNA methylation such as with immunoprecipitation or bisulfite sequencing, chromatin analysis with chromatin immunoprecipitation (ChIP) combined with qPCR or sequencing, and RNA modification study. As researchers elucidate the mechanisms and specific modifications associated with cancer, it may be possible to target abnormal modifications in cells with minimal damage to normal cells, making the prospect of epigenetic therapy increasingly promising.</p>
<h3>Diagenode products for your epigenomics research in Cancer</h3>
<div class="row">
<div class="small-12 medium-4 large-4 columns text-left">
<div class="panel" style="border-color: #099f92; height: 275px;">
<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/chromatin-function">Chromatin analysis</a></h3>
<center><a href="https://www.diagenode.com/en/categories/chromatin-function"><img src="https://www.diagenode.com/img/cancer/chromatin-icon.png" /></a></center>
<p class="text-left">Understand the role of chromatin in cancer regulation</p>
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<li>New to ChIP? <a href="https://www.diagenode.com/en/pages/portal/chip">Learn more</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-shearing">Chromatin shearing optimization</a></li>
<li><a href="https://www.diagenode.com/en/categories/chromatin-immunoprecipitation">Chromatin Immunoprecipitation</a></li>
<li>Validated <a href="https://www.diagenode.com/en/categories/antibodies">Antibodies</a></li>
<li><a href="https://www.diagenode.com/en/categories/library-preparation">Library preparation</a></li>
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<h3 class="text-center"><a href="https://www.diagenode.com/en/categories/dna-methylation" style="color: #30415c;">DNA methylation</a></h3>
<center><a href="https://www.diagenode.com/en/categories/dna-methylation"><img src="https://www.diagenode.com/img/cancer/dna-icon.png" /></a></center>
<p class="text-left">Analyze DNA methylation and the effects on oncogenesis and tumor suppression</p>
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<li><a href="https://www.diagenode.com/en/categories/methylated-dna-immunoprecipitation">Methylated DNA immunoprecipitation</a></li>
<li><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">DNA methylation services</a></li>
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<h3 class="text-center"><span class="darkgrey">Non-coding RNAs</span></h3>
<center><img src="https://www.diagenode.com/img/cancer/non-coding-icon.png" /></center>
<p class="text-left">Discover noncoding RNAs in cancer</p>
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<li><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">RNA-seq using D-Plex</a> </li>
<li><a href="https://www.diagenode.com/en/categories/rna-seq-category">RNA-seq services</a></li>
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<p><strong><input name="data[Cart][quantity]" placeholder="1" value="1" min="1" style="width:60px;display:inline" type="number" id="CartQuantity" required="required"/></strong>Premium RRBS kit V2 <br /> RRBS for low DNA amounts and accurate analysis個カートに追加。</p>
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<h6 style="height:60px">Premium RRBS kit V2</h6>
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<h3>Get a quote</h3><p class="lead">You are about to request a quote for <strong>our epigenomics services</strong>. Fill out the form below and we will be in touch with you very soon.</p><p><small>All <span style="font-size:16px;color:red;">*</span> fields are mandatory</small></p>
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<h2>Service Information</h2>
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<div class="small-12 large-12 columns">
<h4>Which services are you interested in?</h4>
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<div class="small-12 large-12 columns">
<input type="hidden" name="data[Quote][epigenomics_service]" value="" id="QuoteEpigenomicsService"/>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="ChIP-seq" id="QuoteEpigenomicsServiceChIPSeq" /><label for="QuoteEpigenomicsServiceChIPSeq">ChIP-seq</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="ATAC-seq" id="QuoteEpigenomicsServiceATACSeq" /><label for="QuoteEpigenomicsServiceATACSeq">ATAC-seq</label></div>
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<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="WGBS" id="QuoteEpigenomicsServiceWGBS" /><label for="QuoteEpigenomicsServiceWGBS">WGBS</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="MeDIP-seq" id="QuoteEpigenomicsServiceMeDIPSeq" /><label for="QuoteEpigenomicsServiceMeDIPSeq">MeDIP-seq</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Targeted DNA methylation analysis" id="QuoteEpigenomicsServiceTargetedDNAMethylationAnalysis" /><label for="QuoteEpigenomicsServiceTargetedDNAMethylationAnalysis">Targeted DNA methylation analysis</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Infinium MethylationEPIC Array v2" id="QuoteEpigenomicsServiceInfiniumMethylationEPICArrayV2" /><label for="QuoteEpigenomicsServiceInfiniumMethylationEPICArrayV2">Infinium MethylationEPIC Array v2</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Infinium Mouse Methylation Array" id="QuoteEpigenomicsServiceInfiniumMouseMethylationArray" /><label for="QuoteEpigenomicsServiceInfiniumMouseMethylationArray">Infinium Mouse Methylation Array</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="RNA-seq" id="QuoteEpigenomicsServiceRNASeq" /><label for="QuoteEpigenomicsServiceRNASeq">RNA-seq</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Bioinformatics" id="QuoteEpigenomicsServiceBioinformatics" /><label for="QuoteEpigenomicsServiceBioinformatics">Bioinformatics</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Data mining" id="QuoteEpigenomicsServiceDataMining" /><label for="QuoteEpigenomicsServiceDataMining">Data mining</label></div>
<div class="checkbox"><input type="checkbox" name="data[Quote][epigenomics_service][]" value="Human Methylome" id="QuoteEpigenomicsServiceHumanMethylome" /><label for="QuoteEpigenomicsServiceHumanMethylome">Human Methylome</label></div>
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<div class="row collapse">
<div class="small-12 medium-12 large-3 columns">
<span class="prefix">Sample species</span>
</div>
<div class="small-12 medium-12 large-9 columns">
<input name="data[Quote][sample_species]" maxlength="510" type="text" id="QuoteSampleSpecies"/> </div>
</div>
<div class="row collapse">
<div class="small-12 medium-12 large-6 columns">
<span class="prefix">Total number of samples (including replicates)</span>
</div>
<div class="small-12 medium-12 large-6 columns">
<input name="data[Quote][number_samples]" maxlength="255" type="text" id="QuoteNumberSamples"/> </div>
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<div class="row collapse">
<h2>Contact Information</h2>
<div class="small-3 large-2 columns">
<span class="prefix">First name <sup style="font-size:16px;color:red;">*</sup></span>
</div>
<div class="small-9 large-10 columns">
<input name="data[Quote][first_name]" placeholder="john" maxlength="255" type="text" id="QuoteFirstName" required="required"/> </div>
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<span class="prefix">Last name <sup style="font-size:16px;color:red;">*</sup></span>
</div>
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<input name="data[Quote][last_name]" placeholder="doe" maxlength="255" type="text" id="QuoteLastName" required="required"/> </div>
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<span class="prefix">Company <sup style="font-size:16px;color:red;">*</sup></span>
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<input name="data[Quote][company]" placeholder="Organisation / Institute" maxlength="255" type="text" id="QuoteCompany" required="required"/> </div>
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<span class="prefix">Phone number</span>
</div>
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<input name="data[Quote][phone_number]" placeholder="+1 862 209-4680" maxlength="255" type="text" id="QuotePhoneNumber"/> </div>
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<span class="prefix">City</span>
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<option value="AF">Afghanistan</option>
<option value="AX">Åland Islands</option>
<option value="AL">Albania</option>
<option value="DZ">Algeria</option>
<option value="AS">American Samoa</option>
<option value="AD">Andorra</option>
<option value="AO">Angola</option>
<option value="AI">Anguilla</option>
<option value="AQ">Antarctica</option>
<option value="AG">Antigua and Barbuda</option>
<option value="AR">Argentina</option>
<option value="AM">Armenia</option>
<option value="AW">Aruba</option>
<option value="AU">Australia</option>
<option value="AT">Austria</option>
<option value="AZ">Azerbaijan</option>
<option value="BS">Bahamas</option>
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<option value="BD">Bangladesh</option>
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<option value="BA">Bosnia and Herzegovina</option>
<option value="BW">Botswana</option>
<option value="BV">Bouvet Island</option>
<option value="BR">Brazil</option>
<option value="IO">British Indian Ocean Territory</option>
<option value="BN">Brunei Darussalam</option>
<option value="BG">Bulgaria</option>
<option value="BF">Burkina Faso</option>
<option value="BI">Burundi</option>
<option value="KH">Cambodia</option>
<option value="CM">Cameroon</option>
<option value="CA">Canada</option>
<option value="CV">Cape Verde</option>
<option value="KY">Cayman Islands</option>
<option value="CF">Central African Republic</option>
<option value="TD">Chad</option>
<option value="CL">Chile</option>
<option value="CN">China</option>
<option value="CX">Christmas Island</option>
<option value="CC">Cocos (Keeling) Islands</option>
<option value="CO">Colombia</option>
<option value="KM">Comoros</option>
<option value="CG">Congo</option>
<option value="CD">Congo, The Democratic Republic of the</option>
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<option value="CR">Costa Rica</option>
<option value="CI">Côte d'Ivoire</option>
<option value="HR">Croatia</option>
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<option value="CW">Curaçao</option>
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<option value="DJ">Djibouti</option>
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<option value="DO">Dominican Republic</option>
<option value="EC">Ecuador</option>
<option value="EG">Egypt</option>
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<option value="GQ">Equatorial Guinea</option>
<option value="ER">Eritrea</option>
<option value="EE">Estonia</option>
<option value="ET">Ethiopia</option>
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<option value="FO">Faroe Islands</option>
<option value="FJ">Fiji</option>
<option value="FI">Finland</option>
<option value="FR">France</option>
<option value="GF">French Guiana</option>
<option value="PF">French Polynesia</option>
<option value="TF">French Southern Territories</option>
<option value="GA">Gabon</option>
<option value="GM">Gambia</option>
<option value="GE">Georgia</option>
<option value="DE">Germany</option>
<option value="GH">Ghana</option>
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<option value="GR">Greece</option>
<option value="GL">Greenland</option>
<option value="GD">Grenada</option>
<option value="GP">Guadeloupe</option>
<option value="GU">Guam</option>
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<option value="PY">Paraguay</option>
<option value="PE">Peru</option>
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<option value="PT">Portugal</option>
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<option value="MF">Saint Martin (French part)</option>
<option value="PM">Saint Pierre and Miquelon</option>
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<option value="SN">Senegal</option>
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