title: GSM5639710: A549_IFNγ_IRF1_1; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP, cells were grown and fixed with 1% methanol-free formaldehyde (Thermo Scientific) for 10 min and quenched by 125 mmol/L glycine for 15 min at room temperature and, after that, washed with ice-cold PBS twice. Then, the cells were centrifuged (200g at 4°C) for 5 min and the pellet resuspended in 1 mL of lysis buffer (10 mmol/L Tris-HCl, 10 mmol/L NaCl, 0.5% NP-40) containing a Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific; Ref: 78440) and kept rotating for 30 min at 4°C. After centrifugation, the pellet was then 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 a size of 0.25–1.00 kb on average, then frozen at -20ºC for 30 min, 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. Cat: #16-663) 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.