title: GSM8862082: HAP1, SETX WT, DRIP(REP2); Homo sapiens; ChIP-Seq
library_name: GSM8862082
library_construction_protocol: Genomic DNA was extracted from 5–8 million cells per replicate using SDS and proteinase K digestion, followed by phenol/chloroform/ethanol purification. DNA was digested overnight with XbaI, EcoRI, BsrGI, HindIII, and SspI in rCutSmart buffer. RNase H treatment was used as a negative control to degrade DNA-RNA hybrids. Post-cleanup, 12 μg of digested DNA per sample was incubated with 30 μL of S9.6 antibody to capture R-loops. Immunoprecipitation was performed by overnight rotation at 4°C, followed by binding to protein A/G beads for 2.5–4 hours. Bound DNA was eluted using proteinase K digestion at 55°C for 45 minutes, purified via phenol/chloroform/ethanol extraction, and resuspended in 10 mM Tris-HCl (pH 8.0). DNA was stored at -80°C until sequencing. Library preparation was performed using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina following the manufacturer's instructions.