GSM8155648: MED1 ChIP, +DEX +BRM014, BT549 cells, Biorep1; Homo sapiens; ChIP-Seq
For each chromatin immunoprecipitation, cells from two 15cm plates were crosslinked at RT using 2mM disuccinimidyl glutarate (DSG) for 20min followed by 1% formaldehyde for 10min, which was quenched in 0.1M glycine for 10min. Cells were washed twice in cold PBS and scraped off in cold PBS containing protease inhibitors (Roche, Complete 11836170001 and Halt, Thermo Scientific, 78438). Crosslinked material was resuspended in lysis buffer 1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10min followed by 5min in lysis buffer 2 (10mM Tris-HCL pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) before resuspending in lysis buffer 3 (10mM Tris-HCL pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0,5% N-lauroylsarcosine). Samples were sonicated using Covaris Ultrasonicator ME220 until DNA fragments had a size between 150-500bp. Chromatin was immunoprecipitated overnight using protein A coated dynabeads (Dynabeads, 10002D) with specific antibodies. After washing the beads, chromatin was eluted and decrosslinked by incubating at 65 degrees over-night in elution buffer (50mM Tris-HCL, 10mM EDTA, 1% SDS). Samples were treated with RNase A (20ng/mL) for 1h followed by proteinase K (200ng/mL) for 2h before DNA was purified by standard phenol-chloroform purification. Purified DNA was submitted to library preparation using NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E765). The samples were sequenced on a NovaSeq 6000 (Illumi-na) to reach approximately 25M paired-end reads per sample.