GSM5133639: H69_K27Me3; Homo sapiens; ChIP-Seq
Briefly, cells were crosslinked with 1% formaldehyde for 10 min, washed in 5 mg/ml bovine serum albumin (BSA) before re-suspended in lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail) and sonicated with the Covaris M220 sonicator to obtain chromatin fragment lengths of 100-to-1,000 bp. Fragmented chromatin was diluted in IP buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and incubated with Protein G magnetic beads (Dynabeads, Thermo Fisher Scientific, Waltham, MA) pre-incubated with anti-H3K27Ac (ab4729, Abcam, Cambridge, UK) or anti-H3K27me3 (clone C36B1, Cell Signaling Technologies, Danvers, MA) antibodies. Immunoprecipitates were washed six times with wash buffer (50 mM HEPES pH 7.6, 0.5 M LiCl, 1 mM EDTA, 0.7% Na deoxycholate, 1% IGEPAL CA-630) and twice with TE buffer. Immunoprecipitated (or no IP input) DNA was treated with RNase A and Proteinase K on the beads, and recovered in 1% SDS and 0.1 M NaHCO3. Up to 10 ng of DNA was used for the library construction using NEBNext Ultra II DNA Library Prep Kit (Cat# E7645, New England Biolabs, Ipswich, MA). Sequencing was performed on NextSeq500 (Illumina, San Diego, CA) for 38 nucleotides each from paired ends according to the manufacturer's instructions.