Rnase h1

Total genomic nucleic acids were purified by lysing cells with standard Proteinase K/SDS/Tris-EDTA lysis buffer followed by standard phenol/chloroform/isoamyl alcohol pH 8.0 extraction and ethanol precipitation. Samples were digested overnight using a restriction enzyme cocktail of BsrgI, EcoRI, HindIII, SspI, and XbaI in Buffer r2.1 (NEB), followed by a 30-minute incubation with RNase A (Thermo Scientific) in 500mM NaCl to remove free RNA. Genomic DNA was extracted as before and precipitated in isopropanol/NaOAc. As a negative control, 8 μg of each sample was treated with RNase H1 (NEB) at 37°C overnight and then purified as before. 500ng of each sample was spotted onto two wells of positively charged Nylon membrane (Roche) using a slot blot apparatus (BioRad) and vacuum suction. As a separate loading control, half of the membrane was denatured in 0.5M NaOH, 1.5M NaCl, then neutralized for 10 minutes in 1M NaCl, 0.5M Tris-HCl pH 7.0. Membranes were UV crosslinked (0.12J/m²) and then blocked in 5% milk/TBST. Membranes were incubated overnight with either S9.6 (Kerafast #ENH001) or ssDNA (Millipore #MAB3868) antibodies, then washed and incubated with anti-mouse secondary-HRP antibodies (Cell Signaling, #7076) for 1 hour at RT before exposure to ECL.

Genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen) following manufacturer’s protocol. After quantification, different concentrations of DNA (300 ng or 600 ng) were incubated with 1:200 dilutions of RNase T1 (ThermoFisher), RNase III (New England Biolabs), and/or RNaseH1 (New England Biolabs) for 1 h at 37°C to avoid S9.6 antibody artifacts.^{50 (link)} 200 μL DNA solution was loaded to Hybond N+ nylon membrane (GE Life Sciences) presoaked with ddH₂O using the Bio-Dot SF Microfiltration Apparatus (Bio-Rad). The membrane was then crosslinked in the UV Stratalinker 2400 (Stratagene) at the ‘‘Auto Crosslink’’ setting (1200 mJx1000) and incubated with Methylene Blue as a loading control. The membrane was washed in ddH₂O, blocked in 10% milk in TBS-T for 1 h and incubated with S9.6 antibody diluted in TBST-T 2% milk overnight at 4°C to detect RNA:DNA hybrids. Membranes were washed three times in TBS-T and then incubated at room temperature for 1 h with secondary antibody in TBS-T 2% milk. Finally, membranes were washed three times in TBS-T and developed by chemiluminescence (Supersignal West Femto kit, Pierce). Fiji software^{49 (link)} was used to quantify intensity of bands in immunoblots.