title: GSM9386726: 427 cells, control, RNaseH1 overexpression, irradiated; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were washed once with phosphate-buffered saline (PBS; Thermo Fisher) and then pelleted and suspended in 1.6 mL of Tris-EDTA buffer (TE buffer, 10 mM Tris-HCl pH 8, and 1 mM EDTA). 50 µL of SDS 20% (wt./vol) (Santa Cruz) and 5 µL of Proteinase K (20 mg/mL; Invitrogen) were added. Samples were incubated at 37°C overnight. Samples were transferred to 15 mL high-density Maxtract phase lock gel tubes (Qiagen) and one volume (1.6 mL) of Phenol:Chloroform:Isoamyl alcohol (25:24:1; Sigma-Aldrich) was added to each sample. Samples were centrifuged at 1,500 g for 5 min and the supernatant was transferred to a new 15 mL tube. Nucleic acids were precipitated by adding 1/10 volume of 3 M NaOAc pH 5.2 (160 µL) and 2.5 volumes of 100% ethanol (4 mL). Tubes were gently inverted until DNA fully precipitated and became visible. Precipitate was then transferred to a 1.5 mL tube, washed twice with 80% ethanol, air-dried, and resolved in 100 µL of TE buffer. DNA was then fragmented with restriction enzymes (BsrG I, EcoR I, Hind III, Ssp I, Xba I, 30 units each) (NEB) at 37°C overnight. The digested nucleic acids were then recovered by phenol-chloroform extraction. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina following the manufacturer's instructions. 10ng of DNA was used as starting material for input and ip. Libraries were size selected using AMPure XP beads from Beckman Coulter.