GSM8335693: 22Rv1, H3K27me3 ChIPseq, H1-10 OE; Homo sapiens; ChIP-Seq
Cross-linked cells by 1% warm formaldehyde were lysed with 10 ml of LB1 buffer (1 mM EDTA, 50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 0.5% IGEPAL CA-630, 10% Glycerol, 0.25% Triton X-100) on ice for 10 min. Nuclear fractions were centrifugated and resuspended in 10 ml of LB2 buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA, 200 mM NaCl, 0.5 mM EGTA) for 5 min. Nuclear fractions were centrifugated again and resuspended in LB3 buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1×Protease inhibitor cocktail), following with sonication in sonicator (Diagenode bioruptor) to generate 300-700  bp DNA fragments. The sonicated fragments were added to the antibody-conjugated beads and incubated at 4 °C overnight. The beads were washed 10 times with RIPA buffer (50 mM Tris (pH 7.6), 1 mM EDTA, 1 M NaCl, 0.1% SDS, 1% Igepal CA-630, 0.5% sodium deoxycholate) and resuspended in elution buffer (0.1 M NaHCO3, 1% SDS, proteinase K) at 65 °C for 8-16 h to reverse cross-linking of DNA-protein complexes. DNA was purified using the ChIP DNA Clean & Concentrator kit (D5205, Zymo Reasearch). For the ChIP-seq experiments, libraries were prepared with the ThruPLEX DNA-seq kit (Rubicon Genomics).