Successful implementation of ChIP-seq Quality Control at Diagenode using automated ChIP protocol on the SX-8G IP-Star® Compact
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Successful implementation of ChIP-seq Quality Control at Diagenode using automated ChIP protocol on the SX-8G IP-Star® Compact ^ Jan Hendrickx, Geraldine Goens, Catherine D'andrea, Ignacio Mazon, Geoffrey Berguet, Sharon Squazzo, Celine Sabatel, Miklos Laczik, Dominique Poncelet. Diagenode sa, CHU, Tour GIGA B34, 3eme etage 1 Avenue de L'Hopital, 4000 Liege, Sart-Tilman, Belgium Chromatin immunoprecipitation (ChIP) is the most widely used method to study protein-DNA interactions. A successful ChIP, however, is largely depending on the use of well characterised, highly specific ChIP-grade antibodies. We have established a robust QC procedure to be able to provide researchers with the highest quality ChIP-grade antibodies. The recent development of high throughput sequencing (HTS) technologies has also opened new possibilities for epigenetic research. ChIP followed by HTS (ChIP-seq) enables the extension of the target directed analysis of ChIP results to a genome wide analysis. In view of the enormous potential and growing interest of this new development and to increase the quality standards of our antibodies, we added this new technique to our QC procedure. Here, we present 19 ChIP-seq grade antibodies that have passed this very strict quality control procedure. We have validated 19 of the Diagenode ChIP-grade antibodies using ChIP-seq (see table 1). Figure 1 shows the profiles obtained with antibodies against (1) H3K4me3, H3K9ac and H3K9/14ac, histone modifications that are present at active promoters, (2) H3K4me2 which is surrounding promoters of active genes, (3) H3K79me3 present at promoters of active genes and extending into the gene body, and (4) H3K36me3, present at the gene bodies of active genes. The screenshots below show the peak distribution along the complete X-chromosome and in a 75 kb region surrounding the GAPDH gene. Figure 2, 3 and 4 show the profiles of H3K27me3, H3K36me3 and H3K4me3 in a 500 kb region surrounding the MYT1 gene (figure 2), of H3K9me3 in a 200 kb region surrounding ZNF12 (figure 3), and of PolII in a 75 kb region containing the GAPDH gene (figure 3). HeLaS3 were grown under standard conditions, harvested by trypsinisation and fixed for 8 minutes at room temperature with 1% formaldehyde. After washing of the cells with PBS, chromatin was sheared using the shearing ChIP kit ( kch-redmod-100). ChIP was performed on sheared chromatin from 1 million cells with the "Auto Histone ChIP-seq kit protein A" (Cat. No. AB-Auto02-A100) on the SX-8G IP-Star® Automated System using 1 - 2 |jg of the respective antibody. IgG (2 pg/IP) was used as a negative IP control. Depending on the antibody, the IP'd DNA from up to 5 different ChIP reactions was pooled and purified. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer IIx. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the ELAND or BWA algorithm. Diagenode automated ChIP-seq workflow: overview Chromatin stabilized by cross-linking LM.MLiil.jyil, 1.- ill.i, I u I ilLiil. . i, ii.ii i linlii... i.uiiA ii mi n il i H»flnrannmii^ As part of our philosophy to apply the highest quality standards to our antibodies and in an effort to continuously improve our QC procedure, we have introduced the validation of the Diagenode antibodies in ChIP-seq. Currently, a total of 19 ChIP-seq grade antibodies, available at Diagenode, have passed this very strict quality control procedure. Chromatin fragmentation using Bioruptor® UCD series Bioinformatics (llumina pipeline software, ELAND/BWA & algorithms) ER monoclonal antibody TBP monoclonal antibody RARA polyclonal antibody GR monoclonal antibo Human c-fos promoter primer air Human TSH2B primer pair Human myoglobin exon 2 primer pair SX-8G IP-Star® Automated System SX-8G IP-Star® Compact Automated System Auto Histone ChIP-se kit For more information, please contact: for Europe, info@diagenode.com & for the US and Canada, contact info.n