DRIB Protocol for R-loop Detection

The following DRIB protocol was followed to detect R-loops (47 (link), 48 (link)). The nucleus was isolated by hypotonic homogenization. Next, the nucleus was lysed by lysis buffer (1% SDS, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 50 μg/ml Proteinase K) at 55 °C for 2 h. This was followed by phenol: chloroform extraction and 20 μg/ml RNaseA (sigma) treatment for 30 min at 37 °C. The genomic DNA prepared was sonicated in Tris-HCl pH 7.5 with 100 mM NaCl buffer in a probe sonicator thrice for 30 s each in 20% amplitude to yield a maximum fragment size of around ∼1000 bps. For further nucleic acid fragmentation a cocktail of restriction enzymes and purified by phenol: chloroform extraction. This fragmented nucleic acid was transferred to Hybond N membrane (Merck) using a slot blot system and DNA: RNA hybrid was detected by S9.6 antibody (Merck). dsDNA was detected using anti-dsDNA antibody (abcam). Digestion of DNA: RNA hybrid was carried out using RNaseH (NEB).