GSM7989697: PA1_DRIP_RNaseH_minus_rep1; Homo sapiens; RNA-Seq
Genomic DNAs (containing RNAs in the hybrid state) were extracted by phenol/chloroform extraction and ethanol precipitation gently. Then the extracted DNAs were fragmented by treatment with restriction enzymes cocktail (HindIII, EcoRI, BsrGI, XbaI, and SspI) overnight. Fragmented DNAs were recovered by phenol/chloroform extraction and ethanol precipitation gently, and dissolved into 200 μL water. Half of recovered DNAs were treated with RNase H enzyme (Thermo) to eliminate RNAs in the hybrids overnight at 37 ℃ as DRIP control sample, while the remaining half were treated with water as DRIP sample. Digested DNAs were extracted by phenol/chloroform extraction and ethanol precipitation gently, and dissolved into 100 μL water. 10 μg total DNAs in the DRIP sample or DRIP control sample was used for immunoprecipitation (IP) using 5μg S9.6 antibody (Karafast) overnight at 4 ℃, followed by incubation with 50 μL Dynabeads Protein G. The beads were then washed two times with binding buffer (100 mM NaPO4 pH 7.0, 1.4 M NaCl, 0.5% Triton X-100) and eluted by elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) with Proteinase K at 55 ℃ for 1hr. At last, DNAs in the DNA:RNA hybrids were extracted by phenol/chloroform extraction and ethanol precipitation for qPCR. For DRIP-seq, extracted DNAs were further digested with 0.1 mg/mL RNase A at 37 ℃ for 1 hr and recovered by phenol/chloroform extraction and ethanol precipitation. Ribominus RNA libraries were prepared using Illumina TruSeq Stranded Total RNA LT Sample Prep Kit. All libraries were size-selected with AmpureXP beads (Agencourt) and quantified. using Agilent Bio analyzer 2100 and Qubit high-sensitivity dsDNA assay. Size-selected libraries were subjected to deep sequencing with Illumina NextSeq 500 1×100bp single-end Ribominus RNA-seq