title: GSM7627137: WTF_H3K27ac_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Briefly, primary cholangiocytes were divided into 100,000 cells per tube, and the samples were kept on ice or at 4°C. Cells were lysed in 19 μL lysis buffer (0.5% NP-40, 0.5% Tween-20, 0.1% SDS and cOmplete Protease Inhibitor Cocktail), and then incubated with 19 μL MNase buffer (100 mM Tris-HCl pH 8.0 and 2 mM CaCl2) containing 0.02 U MNase (Sigma, N3755) at 37°C for 5 min. Digestion was terminated by adding 5 μL stop buffer (110 mM Tris-HCl pH 8.0 and 55 mM EDTA). Each sample was incubated with 45 μL 2x RIPA buffer (280 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 5 mM EGTA and cOmplete Protease Inhibitor Cocktail) for 10 min before centrifuged at 14500 rpm for 15 min. The supernatant was transferred to a new tube, and then added with 40 μL RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1 mM EDTA). For IP samples, each sample was incubated overnight with 1 μg antibody of H3K9ac (Abcam, ab4441), H3K9me3 (Abcam, ab8898), H3K4me3 (Millipore, 05-745R), H3K4me1 (Abcam, ab8895) or H3K27ac (Abcam, ab4729), and followed by immunoprecipitation with Dynabeads Protein G (Thermo Fisher Scientific, 10004D). And then samples were centrifuged and the collected-beads were washed five times with 150 μL RIPA buffer and rinsed once with 150 μL LiCl buffer (10 mM Tris-HCl pH 8.0, 0.5% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate and 250 mM LiCl). Beads were finally resuspended with 30 μL elution buffer containing 1 μL proteinase K (TransGen Biotech, GE201-01), and followed by shaking at 55°C for 90 min. The supernatant was transferred to a new tube, and proteinase K was inactivated by heated at 72°C for 40 min. For Input samples, each sample was treated with 1 μL proteinase K, followed by shaking at 55°C for 90 min, and then DNA was purified using DNA Extraction Reagent (Solarbio, P1012). Library construction was performed using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607). Sequencing was performed on the Illumina platform.