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<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<div class="large-12 columns">Chromatin Immunoprecipitation (ChIP) coupled with high-throughput massively parallel sequencing as a detection method (ChIP-seq) has become one of the primary methods for epigenomics researchers, namely to investigate protein-DNA interaction on a genome-wide scale. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.</div>
<div class="large-12 columns"></div>
<h5 class="large-12 columns"><strong></strong></h5>
<h5 class="large-12 columns"><strong>The ChIP-seq workflow</strong></h5>
<div class="small-12 medium-12 large-12 columns text-center"><br /><img src="https://www.diagenode.com/img/chip-seq-diagram.png" /></div>
<div class="large-12 columns"><br />
<ol>
<li class="large-12 columns"><strong>Chromatin preparation: </strong>Crosslink chromatin-bound proteins (histones or transcription factors) to DNA followed by cell lysis.</li>
<li class="large-12 columns"><strong>Chromatin shearing:</strong> Fragment chromatin by sonication to desired fragment size (100-500 bp)</li>
<li class="large-12 columns"><strong>Chromatin IP</strong>: Capture protein-DNA complexes with <strong><a href="../categories/chip-seq-grade-antibodies">specific ChIP-seq grade antibodies</a></strong> against the histone or transcription factor of interest</li>
<li class="large-12 columns"><strong>DNA purification</strong>: Reverse cross-links, elute, and purify </li>
<li class="large-12 columns"><strong>NGS Library Preparation</strong>: Ligate adapters and amplify IP'd material</li>
<li class="large-12 columns"><strong>Bioinformatic analysis</strong>: Perform r<span style="font-weight: 400;">ead filtering and trimming</span>, r<span style="font-weight: 400;">ead specific alignment, enrichment specific peak calling, QC metrics, multi-sample cross-comparison etc. </span></li>
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<div class="row" style="margin-top: 32px;">
<div class="small-12 medium-10 large-9 small-centered columns">
<div class="radius panel" style="background-color: #fff;">
<h3 class="text-center" style="color: #b21329;">Need guidance?</h3>
<p class="text-justify">Choose our full ChIP kits or simply choose what you need from antibodies, buffers, beads, chromatin shearing and purification reagents. With the ChIP Kit Customizer, you have complete flexibility on which components you want from our validated ChIP kits.</p>
<div class="row">
<div class="small-6 medium-6 large-6 columns"><a href="../pages/which-kit-to-choose"><img alt="" src="https://www.diagenode.com/img/banners/banner-decide.png" /></a></div>
<div class="small-6 medium-6 large-6 columns"><a href="../pages/chip-kit-customizer-1"><img alt="" src="https://www.diagenode.com/img/banners/banner-customizer.png" /></a></div>
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<div class="large-12 columns">MBD方法は、メチル化DNAに対するH6-GST-MBD融合タンパク質の非常に高い親和性に基づいています。 このタンパク質は、N末端His6タグを含むグルタチオン-S-トランスフェラーゼ（GST）とのC末端融合物として、ヒトMeCP2のメチル結合ドメイン（MBD）を含有します。 このH6-GST-MBD融合タンパク質を用いて、メチル化CpGを含むDNAを特異的に単離する事が可能です。<br /><br />DiagenodeのMethylCap®キットは、二本鎖DNAの高濃縮と、メチル化CpG密度の関数における微分分画を可能にします。 分画はサンプルの複雑さを軽減し、次世代のシーケンシングを容易にします。 MethylCapアッセイに先立ち、DNAを最初に抽出し、Picoruptorソニケーターを用いて断片化します。<br />
<h3>概要</h3>
<p class="text-center"><br /><img src="https://www.diagenode.com/img/applications/methyl_binding_domain_overview.jpg" /></p>
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<p>Diagenode’s <a href="https://www.diagenode.com/en/p/methylcap-kit-x48-48-rxns">MethylCap kit</a> enables high enrichment of double-stranded DNA and a differential fractionation in function of the methylated CpG density. Fractionation reduces the complexity of samples and makes subsequent next generation sequencing easier. Prior to the MethylCap assay, DNA is first extracted and sheared using the <a href="https://www.diagenode.com/en/p/bioruptorpico2">Bioruptor® sonication device</a>.</p>
<h2>How it works</h2>
<center><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/methyl_binding_domain_overview.jpg" /></center>
<h3 class="diacol">ADVANTAGES</h3>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> technique</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<div class="layoutArea"><span>The Auto MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. This procedure has been optimized to perform automated immunoprecipitation of chromatin using the </span><a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a><span><span> </span>enabling highly reproducible results and allowing for high throughput.</span></div>
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'description' => '<p>The MethylCap protein has been extensively validated for specific isolation of DNA fragments containing methylated CpGs. It consists of the methyl-CpG-binding domain (MBD) of human MeCP2, as a C-terminal fusion with Glutathione-S-transferase (GST) containing an N-terminal His6-tag. A single fully methylated CpG is sufficient for the interaction between the MethylCap protein and methylated DNA fragments.</p>',
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'name' => 'Genome-wide DNA hypermethylation opposes healing in chronic woundpatients by impairing epithelial-to-mesenchymal transition.',
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'description' => '<p>An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. Unbiased whole-genome methylome was studied in the wound-edge (WE) tissue of chronic wound patients. A total of 4689 differentially methylated regions (DMRs) were identified in chronic WE compared to unwounded (UW) human skin. Hypermethylation was more frequently observed (3661 DMRs) in the chronic WE compared to hypomethylation (1028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial to mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA sequencing analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified four genes, ADAM17, NOTCH, TWIST1 and SMURF1, that functionally represent the EMT pathway. Single-cell RNA sequencing studies identified that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces WE gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the WE was achieved by a tissue nanotransfection (TNT) based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene-silencing represents a productive therapeutic strategy to improve wound closure.</p>',
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'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/35819852/',
'doi' => '10.1172/JCI157279',
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'description' => '<p>BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.</p>',
'date' => '2018-06-27',
'pmid' => 'http://www.pubmed.gov/29945573',
'doi' => '10.1186/s12885-018-4609-x',
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'name' => 'Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.',
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'description' => '<p>The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathways genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.</p>',
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'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27245242',
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'name' => 'Auto MethylCap kit',
'description' => '<p>The Auto MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. This procedure has been optimized to perform automated immunoprecipitation of chromatin using the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a> enabling highly reproducible results and allowing for high throughput.</p>',
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<li><strong>Fast & sensitive capture</strong> of methylated DNA</li>
<li><strong>High capture efficiency</strong></li>
<li><strong>Differential fractionation</strong> of methylated DNA by CpG density (3 eluted fractions)</li>
<li><strong>Automation compatibility</strong><strong></strong>
<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
<p><img src="https://www.diagenode.com/img/product/kits/mbd_results1.png" alt="MBD-sequencing results have been validated by bisulfite sequencing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>F</strong><strong>igure 1.</strong><span> </span>Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).<br /><strong></strong></p>
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<div class="large-12 columns">Chromatin Immunoprecipitation (ChIP) coupled with high-throughput massively parallel sequencing as a detection method (ChIP-seq) has become one of the primary methods for epigenomics researchers, namely to investigate protein-DNA interaction on a genome-wide scale. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.</div>
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<h5 class="large-12 columns"><strong></strong></h5>
<h5 class="large-12 columns"><strong>The ChIP-seq workflow</strong></h5>
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<li class="large-12 columns"><strong>DNA purification</strong>: Reverse cross-links, elute, and purify </li>
<li class="large-12 columns"><strong>NGS Library Preparation</strong>: Ligate adapters and amplify IP'd material</li>
<li class="large-12 columns"><strong>Bioinformatic analysis</strong>: Perform r<span style="font-weight: 400;">ead filtering and trimming</span>, r<span style="font-weight: 400;">ead specific alignment, enrichment specific peak calling, QC metrics, multi-sample cross-comparison etc. </span></li>
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<div class="small-12 medium-10 large-9 small-centered columns">
<div class="radius panel" style="background-color: #fff;">
<h3 class="text-center" style="color: #b21329;">Need guidance?</h3>
<p class="text-justify">Choose our full ChIP kits or simply choose what you need from antibodies, buffers, beads, chromatin shearing and purification reagents. With the ChIP Kit Customizer, you have complete flexibility on which components you want from our validated ChIP kits.</p>
<div class="row">
<div class="small-6 medium-6 large-6 columns"><a href="../pages/which-kit-to-choose"><img alt="" src="https://www.diagenode.com/img/banners/banner-decide.png" /></a></div>
<div class="small-6 medium-6 large-6 columns"><a href="../pages/chip-kit-customizer-1"><img alt="" src="https://www.diagenode.com/img/banners/banner-customizer.png" /></a></div>
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<div class="large-12 columns">MBD方法は、メチル化DNAに対するH6-GST-MBD融合タンパク質の非常に高い親和性に基づいています。 このタンパク質は、N末端His6タグを含むグルタチオン-S-トランスフェラーゼ（GST）とのC末端融合物として、ヒトMeCP2のメチル結合ドメイン（MBD）を含有します。 このH6-GST-MBD融合タンパク質を用いて、メチル化CpGを含むDNAを特異的に単離する事が可能です。<br /><br />DiagenodeのMethylCap®キットは、二本鎖DNAの高濃縮と、メチル化CpG密度の関数における微分分画を可能にします。 分画はサンプルの複雑さを軽減し、次世代のシーケンシングを容易にします。 MethylCapアッセイに先立ち、DNAを最初に抽出し、Picoruptorソニケーターを用いて断片化します。<br />
<h3>概要</h3>
<p class="text-center"><br /><img src="https://www.diagenode.com/img/applications/methyl_binding_domain_overview.jpg" /></p>
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<h2>How it works</h2>
<center><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/methyl_binding_domain_overview.jpg" /></center>
<h3 class="diacol">ADVANTAGES</h3>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> technique</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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'name' => 'Genome-wide DNA hypermethylation opposes healing in chronic woundpatients by impairing epithelial-to-mesenchymal transition.',
'authors' => 'Singh Kanhaiya et al.',
'description' => '<p>An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. Unbiased whole-genome methylome was studied in the wound-edge (WE) tissue of chronic wound patients. A total of 4689 differentially methylated regions (DMRs) were identified in chronic WE compared to unwounded (UW) human skin. Hypermethylation was more frequently observed (3661 DMRs) in the chronic WE compared to hypomethylation (1028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial to mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA sequencing analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified four genes, ADAM17, NOTCH, TWIST1 and SMURF1, that functionally represent the EMT pathway. Single-cell RNA sequencing studies identified that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces WE gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the WE was achieved by a tissue nanotransfection (TNT) based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene-silencing represents a productive therapeutic strategy to improve wound closure.</p>',
'date' => '2022-07-01',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/35819852/',
'doi' => '10.1172/JCI157279',
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'name' => 'Gene promoter and exon DNA methylation changes in colon cancer development - mRNA expression and tumor mutation alterations.',
'authors' => 'Molnár B, Galamb O, Péterfia B, Wichmann B, Csabai I, Bodor A, Kalmár A, Szigeti KA, Barták BK, Nagy ZB, Valcz G, Patai ÁV, Igaz P, Tulassay Z',
'description' => '<p>BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.</p>',
'date' => '2018-06-27',
'pmid' => 'http://www.pubmed.gov/29945573',
'doi' => '10.1186/s12885-018-4609-x',
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'name' => 'Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.',
'authors' => 'Galamb O et al.',
'description' => '<p>The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathways genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.</p>',
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'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27245242',
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<li><strong>Fast & sensitive capture</strong> of methylated DNA</li>
<li><strong>High capture efficiency</strong></li>
<li><strong>Differential fractionation</strong> of methylated DNA by CpG density (3 eluted fractions)</li>
<li><strong>Automation compatibility</strong><strong></strong>
<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
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<p><strong>F</strong><strong>igure 1.</strong><span> </span>Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).<br /><strong></strong></p>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
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<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<div class="large-12 columns">Chromatin Immunoprecipitation (ChIP) coupled with high-throughput massively parallel sequencing as a detection method (ChIP-seq) has become one of the primary methods for epigenomics researchers, namely to investigate protein-DNA interaction on a genome-wide scale. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.</div>
<div class="large-12 columns"></div>
<h5 class="large-12 columns"><strong></strong></h5>
<h5 class="large-12 columns"><strong>The ChIP-seq workflow</strong></h5>
<div class="small-12 medium-12 large-12 columns text-center"><br /><img src="https://www.diagenode.com/img/chip-seq-diagram.png" /></div>
<div class="large-12 columns"><br />
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<li class="large-12 columns"><strong>Chromatin preparation: </strong>Crosslink chromatin-bound proteins (histones or transcription factors) to DNA followed by cell lysis.</li>
<li class="large-12 columns"><strong>Chromatin shearing:</strong> Fragment chromatin by sonication to desired fragment size (100-500 bp)</li>
<li class="large-12 columns"><strong>Chromatin IP</strong>: Capture protein-DNA complexes with <strong><a href="../categories/chip-seq-grade-antibodies">specific ChIP-seq grade antibodies</a></strong> against the histone or transcription factor of interest</li>
<li class="large-12 columns"><strong>DNA purification</strong>: Reverse cross-links, elute, and purify </li>
<li class="large-12 columns"><strong>NGS Library Preparation</strong>: Ligate adapters and amplify IP'd material</li>
<li class="large-12 columns"><strong>Bioinformatic analysis</strong>: Perform r<span style="font-weight: 400;">ead filtering and trimming</span>, r<span style="font-weight: 400;">ead specific alignment, enrichment specific peak calling, QC metrics, multi-sample cross-comparison etc. </span></li>
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<div class="small-12 medium-10 large-9 small-centered columns">
<div class="radius panel" style="background-color: #fff;">
<h3 class="text-center" style="color: #b21329;">Need guidance?</h3>
<p class="text-justify">Choose our full ChIP kits or simply choose what you need from antibodies, buffers, beads, chromatin shearing and purification reagents. With the ChIP Kit Customizer, you have complete flexibility on which components you want from our validated ChIP kits.</p>
<div class="row">
<div class="small-6 medium-6 large-6 columns"><a href="../pages/which-kit-to-choose"><img alt="" src="https://www.diagenode.com/img/banners/banner-decide.png" /></a></div>
<div class="small-6 medium-6 large-6 columns"><a href="../pages/chip-kit-customizer-1"><img alt="" src="https://www.diagenode.com/img/banners/banner-customizer.png" /></a></div>
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<div class="large-12 columns">MBD方法は、メチル化DNAに対するH6-GST-MBD融合タンパク質の非常に高い親和性に基づいています。 このタンパク質は、N末端His6タグを含むグルタチオン-S-トランスフェラーゼ（GST）とのC末端融合物として、ヒトMeCP2のメチル結合ドメイン（MBD）を含有します。 このH6-GST-MBD融合タンパク質を用いて、メチル化CpGを含むDNAを特異的に単離する事が可能です。<br /><br />DiagenodeのMethylCap®キットは、二本鎖DNAの高濃縮と、メチル化CpG密度の関数における微分分画を可能にします。 分画はサンプルの複雑さを軽減し、次世代のシーケンシングを容易にします。 MethylCapアッセイに先立ち、DNAを最初に抽出し、Picoruptorソニケーターを用いて断片化します。<br />
<h3>概要</h3>
<p class="text-center"><br /><img src="https://www.diagenode.com/img/applications/methyl_binding_domain_overview.jpg" /></p>
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<p>Diagenode’s <a href="https://www.diagenode.com/en/p/methylcap-kit-x48-48-rxns">MethylCap kit</a> enables high enrichment of double-stranded DNA and a differential fractionation in function of the methylated CpG density. Fractionation reduces the complexity of samples and makes subsequent next generation sequencing easier. Prior to the MethylCap assay, DNA is first extracted and sheared using the <a href="https://www.diagenode.com/en/p/bioruptorpico2">Bioruptor® sonication device</a>.</p>
<h2>How it works</h2>
<center><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/methyl_binding_domain_overview.jpg" /></center>
<h3 class="diacol">ADVANTAGES</h3>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> technique</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<div class="layoutArea"><span>The Auto MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. This procedure has been optimized to perform automated immunoprecipitation of chromatin using the </span><a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a><span><span> </span>enabling highly reproducible results and allowing for high throughput.</span></div>
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'description' => '<p>An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. Unbiased whole-genome methylome was studied in the wound-edge (WE) tissue of chronic wound patients. A total of 4689 differentially methylated regions (DMRs) were identified in chronic WE compared to unwounded (UW) human skin. Hypermethylation was more frequently observed (3661 DMRs) in the chronic WE compared to hypomethylation (1028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial to mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA sequencing analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified four genes, ADAM17, NOTCH, TWIST1 and SMURF1, that functionally represent the EMT pathway. Single-cell RNA sequencing studies identified that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces WE gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the WE was achieved by a tissue nanotransfection (TNT) based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene-silencing represents a productive therapeutic strategy to improve wound closure.</p>',
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'description' => '<p>BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.</p>',
'date' => '2018-06-27',
'pmid' => 'http://www.pubmed.gov/29945573',
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'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27245242',
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<p>The DNA recovery after ChIP or MeDIP might be an issue for specific downstream applications (e.g. Next generation sequencing). Diagenode's IPure kit is the only DNA purification kit that is specifically optimized for extracting very low amounts of DNA after ChIP & MeDIP.</p>',
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
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<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<div class="large-12 columns">Chromatin Immunoprecipitation (ChIP) coupled with high-throughput massively parallel sequencing as a detection method (ChIP-seq) has become one of the primary methods for epigenomics researchers, namely to investigate protein-DNA interaction on a genome-wide scale. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.</div>
<div class="large-12 columns"></div>
<h5 class="large-12 columns"><strong></strong></h5>
<h5 class="large-12 columns"><strong>The ChIP-seq workflow</strong></h5>
<div class="small-12 medium-12 large-12 columns text-center"><br /><img src="https://www.diagenode.com/img/chip-seq-diagram.png" /></div>
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<li class="large-12 columns"><strong>Chromatin preparation: </strong>Crosslink chromatin-bound proteins (histones or transcription factors) to DNA followed by cell lysis.</li>
<li class="large-12 columns"><strong>Chromatin shearing:</strong> Fragment chromatin by sonication to desired fragment size (100-500 bp)</li>
<li class="large-12 columns"><strong>Chromatin IP</strong>: Capture protein-DNA complexes with <strong><a href="../categories/chip-seq-grade-antibodies">specific ChIP-seq grade antibodies</a></strong> against the histone or transcription factor of interest</li>
<li class="large-12 columns"><strong>DNA purification</strong>: Reverse cross-links, elute, and purify </li>
<li class="large-12 columns"><strong>NGS Library Preparation</strong>: Ligate adapters and amplify IP'd material</li>
<li class="large-12 columns"><strong>Bioinformatic analysis</strong>: Perform r<span style="font-weight: 400;">ead filtering and trimming</span>, r<span style="font-weight: 400;">ead specific alignment, enrichment specific peak calling, QC metrics, multi-sample cross-comparison etc. </span></li>
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<div class="small-12 medium-10 large-9 small-centered columns">
<div class="radius panel" style="background-color: #fff;">
<h3 class="text-center" style="color: #b21329;">Need guidance?</h3>
<p class="text-justify">Choose our full ChIP kits or simply choose what you need from antibodies, buffers, beads, chromatin shearing and purification reagents. With the ChIP Kit Customizer, you have complete flexibility on which components you want from our validated ChIP kits.</p>
<div class="row">
<div class="small-6 medium-6 large-6 columns"><a href="../pages/which-kit-to-choose"><img alt="" src="https://www.diagenode.com/img/banners/banner-decide.png" /></a></div>
<div class="small-6 medium-6 large-6 columns"><a href="../pages/chip-kit-customizer-1"><img alt="" src="https://www.diagenode.com/img/banners/banner-customizer.png" /></a></div>
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<div class="large-12 columns">MBD方法は、メチル化DNAに対するH6-GST-MBD融合タンパク質の非常に高い親和性に基づいています。 このタンパク質は、N末端His6タグを含むグルタチオン-S-トランスフェラーゼ（GST）とのC末端融合物として、ヒトMeCP2のメチル結合ドメイン（MBD）を含有します。 このH6-GST-MBD融合タンパク質を用いて、メチル化CpGを含むDNAを特異的に単離する事が可能です。<br /><br />DiagenodeのMethylCap®キットは、二本鎖DNAの高濃縮と、メチル化CpG密度の関数における微分分画を可能にします。 分画はサンプルの複雑さを軽減し、次世代のシーケンシングを容易にします。 MethylCapアッセイに先立ち、DNAを最初に抽出し、Picoruptorソニケーターを用いて断片化します。<br />
<h3>概要</h3>
<p class="text-center"><br /><img src="https://www.diagenode.com/img/applications/methyl_binding_domain_overview.jpg" /></p>
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<p>Diagenode’s <a href="https://www.diagenode.com/en/p/methylcap-kit-x48-48-rxns">MethylCap kit</a> enables high enrichment of double-stranded DNA and a differential fractionation in function of the methylated CpG density. Fractionation reduces the complexity of samples and makes subsequent next generation sequencing easier. Prior to the MethylCap assay, DNA is first extracted and sheared using the <a href="https://www.diagenode.com/en/p/bioruptorpico2">Bioruptor® sonication device</a>.</p>
<h2>How it works</h2>
<center><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/methyl_binding_domain_overview.jpg" /></center>
<h3 class="diacol">ADVANTAGES</h3>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> technique</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<div class="layoutArea"><span>The Auto MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. This procedure has been optimized to perform automated immunoprecipitation of chromatin using the </span><a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a><span><span> </span>enabling highly reproducible results and allowing for high throughput.</span></div>
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'name' => 'Genome-wide DNA hypermethylation opposes healing in chronic woundpatients by impairing epithelial-to-mesenchymal transition.',
'authors' => 'Singh Kanhaiya et al.',
'description' => '<p>An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. Unbiased whole-genome methylome was studied in the wound-edge (WE) tissue of chronic wound patients. A total of 4689 differentially methylated regions (DMRs) were identified in chronic WE compared to unwounded (UW) human skin. Hypermethylation was more frequently observed (3661 DMRs) in the chronic WE compared to hypomethylation (1028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial to mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA sequencing analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified four genes, ADAM17, NOTCH, TWIST1 and SMURF1, that functionally represent the EMT pathway. Single-cell RNA sequencing studies identified that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces WE gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the WE was achieved by a tissue nanotransfection (TNT) based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene-silencing represents a productive therapeutic strategy to improve wound closure.</p>',
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'description' => '<p>BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.</p>',
'date' => '2018-06-27',
'pmid' => 'http://www.pubmed.gov/29945573',
'doi' => '10.1186/s12885-018-4609-x',
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