GSM9512677: CTRL 2 NS cells DRIP; Homo sapiens; ChIP-Seq
DNA-RNA Immunoprecipitation (DRIP). The same cell lines were used for DRIP as for ChIP. 5 to 7 million cells were harvested for the DRIP experiment, subsequently stored at -70°C. Genomic and mitochondrial DNA were isolated using the Macherey-Nagel NucleoSpin Tissue Kit. We modified the manufacturer's protocol by performing the lysis step at 55°C for 4 hours, instead of the recommended 70°C for 15 minutes, to preserve R-loops from degradation 46. Post-RNase A digestion (37°C, 18 min), DNA concentration was measured. For each sample type, 6 µg of genomic DNA (gDNA) was sonicated for 5 cycles at high intensity (1 cycle: 30s ON, 30s OFF) to achieve fragment sizes of 400-600 bp, verified by agarose gel electrophoresis. We reserved 2% of each sample as INPUT. As a negative control, 6 µg of DNA underwent RNase H digestion in a thermoshaker at 37°C for 2 hours (RNase H selectively cleaves RNA-DNA hybrids). RNase H-digested samples were then purified using the Kapa Pure bead system according to the manufacturer's instructions. For immunoprecipitation, 100 µL of protein G beads were washed twice with 0.5% BSA in PBS/EDTA, then incubated with 30 µg of S9.6 antibody (R-loop specific) in 0.5% BSA PBS/EDTA for 4 hours at 4°C on a rotator. The S9.6-bead complexes were washed twice, then immunoprecipitation was performed in IP buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 5 mM EDTA, 1% Triton-X, 0.1% Na-deoxycholate) containing Protease Inhibitor. The samples were added to the bead-S9.6 complex and incubated overnight at 4°C on a rotator. The next day, bead-S9.6-R-loop complexes were washed thrice with IP buffer and thrice with TE buffer, then resuspended in elution buffer (50 mM Tris/HCl pH 8, 10 mM EDTA, 1% SDS, proteinase K), and incubated at 55°C for 2 hours at 1000 rpm in a thermos shaker. The supernatant was then transferred to a new tube and purified using a 3X bead-to-sample ratio with Kapa Pure beads. NGS library preparation and sequencing was performed using the Illumina TruSeq ChIP Sample Preparation protocol. Briefly, DRIP DNA was end-repaired and indexed adapters were ligated to the inserts. Purified ligation products were then amplified by PCR. Amplified libraries were sequenced