GSM2876325: ChIP-seq_H3K27Ac_resistant_JQ1_treated; Homo sapiens; ChIP-Seq
Cells were harvested, washed twice in ice cold PBS with protease inhibitors and then lysed in 50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS supplemented with protease inhibitors (Roche). Chromatin was sheared to ~250 bp fragments using the Covaris ultra-sonication. The sonicated lysate was then diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X100, 0.01% SDS).  Subsequently, 2.5% of the lysate was removed as the input control.  The remaining lysate was incubated over night at 4°C with 3 µg of H3K27Ac antibody (ab4729, Abcam). The precipitated lysate was then incubated with protein A/G sepharose beads (Pierce, catalog #20423) for 2 hours and then washed sequentially in ice cold low salt buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton-X100, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% deoxycholic acid, 1% IGEPAL CA-630) and TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).  Samples were incubated at 65°C overnight with elution buffer (0.1 M NaHCO3, 1% SDS) supplemented with 0.2M NaCl to reverse crosslinking. ChIP libraries were prepared using the Rubicon Genomics Thruplex DNA-seq prep kit optimized for low input samples.