GSM4593533: KO3; Homo sapiens; ChIP-Seq
Cell pellets biological triplicates/group) were crosslinked with 1% formaldehyde and quenched with 125 mM glycine. Pelleted cells were washed thoroughly with 1XPBS containing protease inhibitors and subjected to lysis and sonication, as previously described (Lee et al. 2006). Briefly, pellets were re-suspended and rotated for 15 minutes in 1 mL LB1 buffer [50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton-X 100 plus protease inhibitors] at 4°C. Following pelleting, cells were then re-suspended in 1 mL LB2 buffer [10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, plus protease inhibitors] and rotated for 10 minutes at 4°C. Pelleted cells/nuclei were then re-suspended in 1 mL LB3 buffer [10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine plus protease inhibitors] and sonicated using a Covaris S220 (Covaris Inc., Worburn, MA, USA) for 50 minutes at 4°C, resulting in approximately 150-300 bp DNA fragments. Following sonication, 1/10 volume of 10% Triton-X 100 was added and samples were spun at 18,000 x g for 10 minutes at 4°C to crack nuclei. Supernatants were then collected and incubated with a rabbit anti-H3K9me3 ChIP-grade antibody (Abcam, ab8898, Lot GR3217595-2, 6 μg/sample) bound sheep anti-rabbit IgG M-280 Dynabeads (Invitrogen, 11204D) on a rotator at 4°C overnight. The following day, immunoprecipitates were washed 8X with 1 mL RIPA buffer [50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate, plus protease inhibitors], rinsed 1X with 1 mL TE + 50 mM NaCl buffer and then eluted at 65°C for 30 minutes (shaking at 1000 RPM) in 210 μl elution buffer [50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS]. After centrifugation, samples, and respective inputs, were reversed crosslinked in elution buffer 65°C overnight. Next, RNA and protein were digested with RNAse (one hour at 37°C; Roche, 10109134001) and proteinase K (two hours at 55°C; Roche, 3115887001), respectively, prior to DNA fragment purification using a Qiagen PCR purification kit (Qiagen, 28106). ChIP-seq libraries were prepared according to Illumina protocols and sequenced on an Illumina HiSeq 4000 Sequencer_High Output mode v4.
GEO
GSM4593533