GSM8776084: NU1 cells, proliferating, STAG2, biol rep 2; Mus musculus; ChIP-Seq
Cells (4 × 107) were harvested and chromatin was crosslinked, quenched, sonicated and bound to protein A/G agarose beads. The supernatant was divided into aliquots (550 μg/assay) and incubated with 25 μg of antibody (STAG1, STAG2, CTCF, non-related IgG) overnight at 4 ºC on a rotating platform. Beads were sequentially washed with low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and TE 1X. DNA was eluted (1% SDS, 0.1 M NaHCO3) and purified by phenol–chloroform extraction once cross-linking was reversed. 5ng of DNA per sample was used as provided by the user. Samples were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters with "NEBNext Ultra II DNA Library Prep Kit for Illumina" from New England BioLabs (catalog # E7645). Adapter-ligated libraries were completed by limited-cycle PCR and extracted with a [single] double-sided SPRI size selection. Resulting average fragment size is 450 bp from which 120 bp correspond to adaptor sequences. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument (see below) by following manufacturer's protocols.