title: GSM8262522: A673 STAG2ko, Input ChIP-Seq; Homo sapiens; ChIP-Seq
library_construction_protocol: A673 cells grown at high confluence were crosslinked with 1% formaldehyde for 15 min at room temperature. After quenching the reaction with 0.125 M Glycine, fixed cells were washed twice with PBS containing 1μM PMSF and protease inhibitors. Cells were lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) at a concentration of 2x107 cells/ml. Sonication was performed with a Covaris S220 (shearing time 20 min, 20% duty cycle, intensity 6, 200 cycles per burst and 30 sec per cycle) in a minimum volume of 2 ml. Chromatin from 107 cells was incubated with 5 μg of antibody as described . For calibration, 5% of chromatin from mouse ES cells was added to the human chromatin For library preparation, at least 5 ng of DNA were processed through subsequent enzymatic treatments with "NEBNext Ultra II FS DNA Library Prep Kit for Illumina" from New England BioLabs (cat# E7805). Briefly, a short fragmentation of 10 min was followed by end-repair, dA-tailing, and ligation to adapters. Adapter-ligated libraries were completed by limited-cycle PCR (8-12 cycles). Resulting average fragment size is 300 bp from which 120 bp correspond to adaptor sequences. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on Illumina NextSeq 500 (with v2.5 reagent kits) following manufacturer's recommendation