title: GSM9076657: BJ sgCtrl-input; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assays were carried out as previously described. Briefly, BJ cells were fixed with 1% formaldehyde (F8775, Sigma) for 10 min at room temperature, and a final concentration of 125 mM glycine was used to quench the reaction. Cells were washed twice with cold PBS and collected in cold PBS by scraping, then resuspended in hypotonic lysis buffer (20 mM Hepes, pH 7.9, with 10 mM KCl, 10% glycerol, 1 mM DTT and a protease inhibitor cocktail (04693124001, Roche)). The pellet of nuclei was resuspended in RIPA buffer (10 mM Tris-HCl, pH 8.0, with 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate and Protease Inhibitor). Nuclear extracts were sonicated to generate chromatin fragment on a Covaris M220 Focused-ultrasonicator (Insert, microTUBE 130 µL; Temperature, 7°C; Peak Incident Power, 75W; Duty Factor, 5%; Treatment Time, 300s). The chromatin was cleaned by centrifugation at 14,000×g for 10 min at 4 °C and diluted with 1 mL RIPA buffer per reaction. Then the chromatin was precleared with Protein G Sepharose 4 Fast Flow (17061801, GE) for 2 hours, 30 µL of sample was set aside as an input and subjected to immunoprecipitation with antibody against H3K9me3 (abcam, ab8898) overnight. On the following day, Pierce Protein A/G Plus Agarose beads (Thermo Fisher Scientific, 20423) that were blocked with RIPA containing 0.5% bovine serum albumin (BSA) were added into the samples followed by incubation for 2 hours in cold room under rocking. The beads were then washed three times with RIPA buffer, followed by two times in RIPA buffer with 0.5 M NaCl, oncein LiCl buffer [250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630,0.1% sodium deoxycholate, and 10 mM tris-HCl (pH 8.0)], and twice in ice-cold Tris- EDTA (TE) buffer. Each time, the beads were sustaining for 5 min with a gentle rock. After washing, both the beads and input sample were added with 150 μL of extraction buffer (1% SDS in 1× TE, 12 μL of 5 M NaCl, and 10 μg of RNAse A) and incubated at 37°C for 1 hour with a shaking. Next, 20 μg of Proteinase K was added to the samples for incubation at 65°C overnight with thermoshaking. DNA was isolated by performing a phenol/chloroform/isoamyl alcohol extraction to prepare the ChIP-seq library using the NEBNext Ultra II DNA Library Prep Kit for Illumi-na (NEB, E7645).