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Successful Implementation of Chip-Seq Quality Control At Successful implementation of ChIP-seq Quality Control at Diagenode using automated ChIP protocol on the SX-8G IP-Star® Compact Jan Hendrickx, Géraldine Goens, Catherine D’andrea, Ignacio Mazon, Geoffrey Berguet, Sharon Squazzo, Céline Sabatel, Miklos Laczik, Dominique Poncelet. Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium Introduction Results Figure 3 ZNF12 Chromatin immunoprecipitation (ChIP) is the most widely used method to study protein-DNA We have validated 19 of the Diagenode ChIP-grade antibodies using ChIP-seq (see table 1). interactions. A successful ChIP, however, is largely depending on the use of well characterised, Scale 100 kb Figure 1 shows the profiles obtained with antibodies against (1) H3K4me3, H3K9ac and H3K9/14ac, chr7: 6620000 6630000 6640000 6650000 6660000 6670000 6680000 6690000 6700000 6710000 6720000 6730000 6740000 6750000 6760000 6770000 6780000 6790000 6800000 highly specific ChIP-grade antibodies. histone modifications that are present at active promoters, (2) H3K4me2 which is surrounding 35 - We have established a robust QC procedure to be able to provide researchers with the highest quality promoters of active genes, (3) H3K79me3 present at promoters of active genes and extending into H3K9me3 ChIP-grade antibodies. The recent development of high throughput sequencing (HTS) technologies the gene body, and (4) H3K36me3, present at the gene bodies of active genes. The screenshots below 1 _ has also opened new possibilities for epigenetic research. ChIP followed by HTS (ChIP-seq) enables show the peak distribution along the complete X-chromosome and in a 75 kb region surrounding the C7orf26 AK123300 ZNF12 PMS2CL RSPH10B C7orf26 ZNF12 RSPH10B the extension of the target directed analysis of ChIP results to a genome wide analysis. GAPDH gene. C7orf26 PMS2CL C7orf28B In view of the enormous potential and growing interest of this new development and to increase the Figure 2, 3 and 4 show the profiles of H3K27me3, H3K36me3 and H3K4me3 in a 500 kb region RSPH10B2 quality standards of our antibodies, we added this new technique to our QC procedure. Here, we surrounding the MYT1 gene (figure 2), of H3K9me3 in a 200 kb region surrounding ZNF12 (figure 3), present 19 ChIP-seq grade antibodies that have passed this very strict quality control procedure. and of PolII in a 75 kb region containing the GAPDH gene (figure 3). Figure 4 GAPDH Methods Figure 1 Scale 20 kb chr12: 6465000 6470000 6475000 6480000 6485000 6490000 6495000 6500000 6505000 6510000 6515000 6520000 6525000 6530000 293 - HeLaS3 were grown under standard conditions, harvested by trypsinisation and fixed for 8 minutes X-chromosome Scale 50 Mb at room temperature with 1% formaldehyde. chrX: 10000000 20000000 30000000 40000000 50000000 60000000 70000000 80000000 90000000 100000000 110000000 120000000 130000000 140000000 150000000 Pol II 422 - H3K4me3 1 _ After washing of the cells with PBS, chromatin was sheared using the shearing ChIP kit ( kch- MRPL51 SCARNA10 GAPDH 1 _ NCAPD2 IFFO1 redmod-100). ChIP was performed on sheared chromatin from 1 million cells with the “Auto Histone 239 - IFFO1 ® IFFO1 ChIP-seq kit protein A” (Cat. No. AB-Auto02-A100) on the SX-8G IP-Star Automated System using H3K9ac IFFO1 1 _ 1 - 2 µg of the respective antibody. IgG (2 µg/IP) was used as a negative IP control. Depending on the 286 - antibody, the IP’d DNA from up to 5 different ChIP reactions was pooled and purified. H3K9/14ac 1 _ 73 - Conclusions 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. H3K4me2 1 _ As part of our philosophy to apply the highest quality standards to our antibodies and in an effort The 36 bp tags were aligned to the human genome using the ELAND or BWA algorithm. 146 - H3K79me3 to continuously improve our QC procedure, we have introduced the validation of the Diagenode 1 _ antibodies in ChIP-seq. Currently, a total of 19 ChIP-seq grade antibodies, available at Diagenode, Diagenode automated ChIP-seq workflow: overview 76 - H3K36me3 have passed this very strict quality control procedure. 1 _ RefSeq Genes Description Cat No. Description Cat No. '$ Hela S3 cells H3K4me3 ER 8]gdbVi^chiVW^a^oZY GADPH pAb-003-050 AC-066-100 polyclonal antibody monoclonal antibody Scale 20 kb WnXgdhh"a^c`^c\ chr12: 6465000 6470000 6475000 6480000 6485000 6490000 6495000 6500000 6505000 6510000 6515000 6520000 6525000 6530000 358 - H3K4me3 TBP MAb-152-050 MAb-002-100 H3K4me3 monoclonal antibody monoclonal antibody 1 _ 217 - H3K4me2 RARA polyclonal pAb-035-050 CS-155-100 H3K9ac polyclonal antibody antibody ($ 1 _ 144 - 8]gdbVi^c[gV\bZciVi^dc H3K4me1 H3K9/14ac pAb-037-050 GR monoclonal antibody MAb-010-050 polyclonal antibody jh^c\7^dgjeidg J89hZg^Zh 1 _ 52 - H3K9me3 Human c-fos promoter 9^?FFheY[ii Shearing ChIP Kit H3K4me2 pAb-056-050 pp-1004-050/500 ® polyclonal antibody primer air Bioruptor Plus (Cat. No. kch-redmod-100) 1 _ 47 - H3K27me3 Human TSH2B H3K79me3 pAb-069-050 pp-1041-050/500 a. b. c. d. 1 _ polyclonal antibody primer pair )$ 39 - 6jidbViZY8]>Edci]Z H3K36me3 Human myoglobin exon 2 HM"-<>E"HiVgjh^c\ H3K36me3 pAb-058-050 pp-1006-050/500 1 _ polyclonal antibody primer pair ChIP-seq grade MRPL51 SCARNA10 GAPDH 6jid8]>E`^i Antibodies NCAPD2 IFFO1 IFFO1 ® IFFO1 H3K36me3 SX-8G IP-Star IFFO1 CS-058-100 UH-001-0001 polyclonal antibody Automated System *$ Auto Histone ChIP-seq Kit* Auto IPure Kit ® HZfjZcX^c\a^WgVgn (Cat. No. AB-Auto02-A100) (Cat. No. AL-Auto01-0100) H3K79me3 SX-8G IP-Star Compact ® pAb-068-050 UH-002-0001 egZeVgVi^dc SX-8G IP-Star polyclonal antibody Automated System Compact Figure 2 H3K9ac ® pAb-004-050 Bioruptor Plus UCD-300-TO +$ MYT1 polyclonal antibody 8]>E"hZfgjcdc<Vaam 7iiWo7dWboi_i >aajb^cVhZfjZcXZg Scale 200 kb H3K9ac Shearing ChIP kit kch-redmod-100 chr20: 62000000 62050000 62100000 62150000 62200000 62250000 62300000 62350000 62400000 pAb-177-050 22 - 8]>E"hZf polyclonal antibody H3K27me3 Shearing ChIP kit kch-redmod-100 H3K9/14ac 1 _ pAb-005-044 ,$ 45 - 8]>E"hZf>ceji9C6 polyclonal antibody 7^d^c[dgbVi^Xhaajb^cV H3K36me3 IPure kit AL-100-0100 H3K27ac e^eZa^cZhd[ilVgZ! 1 _ pAb-174-050 :A6C9$7L6Va\dg^i]bh 306 - polyclonal antibody Auto IPure kit AL-Auto01-0100 :WjW7dWboi_i H3K4me3 1 _ H4K20me3 ABHD16B DNAJC5 SAMD10 LINC00176 TCEA2 NPBWR2 MYT1 LINC00266-1 pAb-057-050 Auto Histone ChIP-seq TPD52L2 UCKL1 PRPF6 TCEA2 PCMTD2 AB-Auto02-A100 TPD52L2 UCKL1 LINC00176 RGS19 PCMTD2 polyclonal antibody TPD52L2 MIR1914 SOX18 RGS19 kit TPD52L2 MIR647 OPRL1 * Alternatively, the iDeal ChIP-seq kit can be used TPD52L2 UCKL1-AS1 OPRL1 Pol II ZNF512B C20orf201 AC-055-100 for performing ChIP manually. OPRL1 monoclonal antibody iDeal ChIP-seq kit x24 AB-001-0024 iDeal ChIP-seq Kit* (Cat. No. AB-001-0024) PO-ChIP-seq-A3-V1_22_02_12 www.diagenode.com For more information, please contact: for Europe, [email protected] & for the US and Canada, contact [email protected].
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