R-loop Immunostaining and Quantification

R-loop immunostaining and fluorescence quantification was performed as described [116 (link)], with the exception of cell lysis, where we mechanically broke cells in liquid nitrogen instead of using enzymatic lysis. R-loops were visualized using ⍺S9.6 antibody [117 (link)]. For negative control samples, we added RNase H (Roche 10786357001) at 3u/100μl and incubated for two hours at 37°C. Nuclei were stained with DAPI at 3μg/mL in 50% glycerol. Images were obtained using a spinning disk confocal microscope (Yokogawa CSU-X1 head mounted on Olympus body), CoolSnap HQ2 camera (Roper Scientific), and a 100X Plan Apochromat 1.4 NA objective (Olympus). Images presented correspond to maximal projections of 10 slides’ stacks using Image J open software [118 (link)].