GSM7765141: U2OS_siHELZ_DRIP_rep1; Homo sapiens; ChIP-Seq
Genomic DNA was extracted from ~2 million U2OS cells and fragmented with restriction enzymes (BsrGI, EcoRI, HindIII, SspI, XbaI, 30 U/each, NEB). Fragmented nucleic acids were recovered by phenol-chloroform extraction. Fragmented gDNA (5 μg) was digested with 20 U of RNase H (NEB #M0297S) for 6 hr to serve as a negative control. Fragmented gDNA or RNase H-digested DNA (5 μg) were immunoprecipitated with 8 μg of S9.6 antibody (Millipore #MABE1095) overnight at 4°C in DRIP binding buffer (10 mM sodium phosphate, 140 mM sodium chloride, 0.05% (v/v) Triton-X-100, pH 7.0). DNA-antibody complex was then incubated with 40 μL of Protein G beads (Invitrogen #10003D) for 2 hr at 4°C, and beads were washed three times with DRIP binding buffer. Antibody-captured DNA-RNA hybrids were then eluted in (50 mM Tris, 10 mM EDTA, 0.5% (v/v) SDS, pH 8.0) and subjected to phenol-chloroform extraction. The immunoprecipitated DNA-RNA hybrids were quantified by Qubit (ThermoFisher Scientific #Q32854). DNA-RNA hybrids (10 ng) were treated with 2 U of RNase H for 1 h and then were sonicated into small fragments (~100-500 bp) using a Covaris Focused-Ultrasonicator Me220. Fragmented DNA was blunted, 5′ phosphorylated and 3' A-tailed by NEBNext Ultra II End Prep Enzyme Mix following the manufacturer's instruction (New England Biolabs #E7645L). An adaptor was ligated, and USER enzyme was used for U excision to yield adaptor-ligated double-strand DNA. The DNA was then PCR-amplified using barcoded PCR primers (New England Biolabs #E7335L, #E7500L). After AMPure XP bead purification (Beckman Coulter #A63881) and Qubit quantification, the libraries were sent to Admera Health for quality analysis and sequencing with Illumina with a read length configuration of 150 PE.