GSM5897810: ChIP ZNF524-GFP WT rep 2; Homo sapiens; ChIP-Seq
Subconfluent U2OS cells were washed twice with cold PBS and cross-linked with 1% formaldehyde (Life Technologies) in DMEM for 20 min at RT. The formaldehyde was quenched with glycine (0.125 M final concentration) for 5 min at RT, cells were scraped with a plastic scraper and washed once in PBS. For lysis, cells were resuspended in lysis buffer 1 (50 mM Tris-HCl pH 8.0, 250 mM sucrose, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5% IGEPAL-630 (Sigma-Aldrich), 0.25% Triton X-100, 0.25% Tween20) for 15 min, pelleted and subsequently resuspended in lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) for 10 min both at 4 ºC. Sonication was performed in 1.5 ml sonication buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0) with a EpiShear probe sonicator (Active Motif)  for 6.25 min (30% amplitude, 15 sec ON, 30 sec OFF) to obtain fragments of 200-400bp. DNA fragments were repaired, A-tailed and then ligated with Illumina adapters. After size selection and PCR amplification, the sequencing library was checked for size distribution using the 2100 Bioanalyzer System (Agilent), quantified using real-time PCR and the Qubit dsDNA HS Assay kit on a Qubit 2.0 fluorometer (Thermo Scientific). The quantified libraries were pooled in equimolar ratio and sequenced on a Illumina NovaSeq 6000.