title: GSM6940137: Control, CTCF, BioRep1; Homo sapiens; ChIP-Seq
library_name: GSM6940137
library_construction_protocol: 5-15 million cells were cross-linked per ChIP experiment in 2mL PBS per 6-well with 1% methanol-free formaldehyde for 5-10 min and quenched with a final concentration of 0.125M glycine for 5 min with nutation. Cross-linked cells were washed with PBS, scraped and pelleted by centrifugation, flash-frozen in liquid nitrogen and stored at -80°C. Samples were defrosted on ice and resuspended in 5mL LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, with 1X cOmplete Protease Inhibitor Cocktail and 1 mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 5mL LB2 (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1X cOmplete Protease Inhibitor Cocktail and 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 1mL LB3 per 10 million cells (up to 1 mL per ChIP). Samples were sonicated in 1mL AFA tubes for 5 min on E220 evolution Covaris with settings Peak power = 140, Duty Factor = 10, Cycles per burst = 200 to achieve chromatin sized approximately 500-2000bp. Following sonication, samples were re-combined (if aliquoted for sonication), Triton X-100 was added to the fragmented chromatin to a final concentration of 1%, and the chromatin divided for input (1%–2%) and ChIP samples. Samples were quantified by Qubit dsDNA HS assay kit, and 30 ng of ChIP DNA was used for library preparation with end repair, A-tailing, and adaptor ligation (NEB). Following USER enzyme treatment, libraries were cleaned up with one round of single-side AMPure XP bead clean-up. Libraries were then amplified to add indices using NEBNext HiFi 2X PCR mix and NEBNext Multiplex Oligos for Illumina kit (NEB, E7335S) with 9 cycles (as determined by qPCR. ChIP libraries were purified by two rounds of double-sided AMPure XP bead clean-up to deplete adaptors. Library concentration and quality within ChIP or input groups was assessed by Qubit dsDNA HS assay kit and used to pool within ChIP or input groups.