GSM5175743: K562 siSAFB&B2 H3K9me3 ChIP-Seq; Homo sapiens; ChIP-Seq
1 million cells were resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 1 mM CaCl2 and protease inhibitor cocktail) and homogenized through a G25 syringe. Chromatin was digested in 0.2 U μl−1 MNase (NEB) at 37 °C for 10 min, and the reaction was quenched by 0.5 M EDTA. Chromatin was resuspended in hypotonic buffer (0.2 mM EDTA, pH 8.0, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 1.5 mM dithiothreitol and 1 × protease inhibitor cocktail (PIC) and incubated for 1 h on ice. Cellular debris was pelleted and the supernatant was recovered. Chromatin was pre-cleared with 20 μl of 1:1 protein A:protein G Dynabeads (Life Technologies) and immunoprecipitation was carried out with antibody–bead complexes (2ug H3K9me3 antibody and 20 μl 1:1 protein A:protein G Dynabeads) overnight at 4 °C. IPed complexes were washed twice with 400 μl of ChIP wash buffer I (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA and 150 mM NaCl) and twice with 400 μl of ChIP wash buffer II (20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 2 mM EDTA and 500 mM NaCl). Protein–DNA complexes were eluted in 200 μl of elution buffer (100 mM NaHCO3 and 1% SDS) for 2 h at 68 °C. IPed material was purified by phenol chloroform and was processed for library construction. DNA libraries were prepared using NEB Next Ultra II DNA library prep kit for illumina according to manufactorer's instructions