R-loop co-immunoprecipitation (co-IP)

Nuclear extracts in RIPA buffer containing 1x Protease inhibitor cocktail (Sigma) were sonicated using Covaris AFA™ (Adaptive Focused Acoustics, Woburn, MA, USA) technology according to the manufacturer’s instructions. After centrifugation (13,000 rpm for 10 min at 4°C), extracts were immunoprecipitated using Protein G Agarose-PLUS (Santa Cruz Biotechnology Inc., TX, USA) pre-coated with S9.6 antibody or control IgG. Following 4 h rotation at 4°C, the beads were washed three times in RIPA buffer. Precipitated R-loops or bound proteins were visualized using immunoblot. For S9.6 dot blot, aliquots of input, IgG and S9.6 IP were treated with proteinase K before subsequent R-loop purification.