GSM9415059: ATRi rep1 S6/G2/M; Homo sapiens; OTHER
Cells were incubated overnight in lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, 0.1% SDS, 50 ug/mL RNAse A, 100 ug/mL Proteinase K). DNA was purified from the lysates by phenol extraction. Four µg of genomic DNA was fragmented to a mean size of 500 bp on a Covaris S220 intrument (peak power: 140, duty factor: 10%, cycle/burst: 200, time: 80 seconds). Fragmented DNA was treated with the End-Repair module (New England BioLabs, E6050) and the A-Tailing module (New England BioLabs, E6053), according to the manufacturer recommendations. Illumina Truseq indexed adapters were ligated on the resulting fragments, using the Quick Ligation module (New England BioLabs, E6056), according to the manufacturer recommendations. Following heat denaturation, BrdU-DNA was isolated by immunoprecipitation using an anti-BrdU monoclonal antibody (BD Biosciences, 347580). Immunoprecipitated fragments were amplified for 10 cycles using the KAPA Hifi DNA polymerase (KAPABiosystems, KK2502) and the resulting libraries were purified with AMPure XP beads (Beckman Coulter, A63881). Illumina libraries were pooled and sequenced on a NextSeq500 instrument on Paired-end 2x43 bp runs, using a NextSeq 500/550 High Output Kit v2 (75 cycles) kit.