GSM4695902: H1299-mutSMARCA4_Dox_SAHA_H3K27Ac; Homo sapiens; ChIP-Seq
For ChIP, cells were grown in P-150 cm cell dishes and fixed with 1%  methanol-free formaldehyde (Thermo Scientific) for 10 min at room temperature, then  quenched by 125 mmol/L glycine for 15 min at room temperature, washed with ice-cold PBS  twice and centrifuged at 200g, 4°C for 5 min. The pellet was resuspended in 1 mL of cell lysis  buffer (10 mmol/L Tris-HCl, 10 mmol/L NaCl, 0.5% NP-40, protease inhibitor) and kept at  4°C, rotating for 30 min. After centrifugation, the pellet was resuspended in 1 mL of nuclear  lysis buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl pH 8.0, protease inhibitor) and  kept at 4°C for 60 min. After another centrifugation, the lysate was sonicated with a Covaris  M220 instrument to yield chromatin fragments of an average size of 0.25–1.00 kb, and then  frozen at -20ºC for 30 min. The chromatin was thawed on ice and centrifuged at 2,500 g. For  each ChIP reaction, 60 μL of Magna ChIP™ Protein A+G Magnetic Beads (Merck Millipore)  was used in accordance with the manufacturer's protocol. Before addition of the sheared  chromatin to the beads, Triton X-100 and Na-deoxycholate was added to a final concentration  of 10% each. 1% of the chromatin volume was used for input. At least two independent ChIP  experiments were performed. Immunoprecipitated chromatin was deep-sequenced in the Genomics Unit of the Centre  for Genomic Regulation (CRG, Barcelona, Spain) using the Illumina HiSeq 2500 system  (Illumina). Briefly, library preparation included end-repair, generation of dA overhangs,  adapter ligation, size selection and removal of non-ligated adapters by agarose gene  electrophoresis and amplification (18 cycles) before loading the samples into the sequencer.