A technique to detect native R-loops genome-wide without immunoprecipitation

We adapted the nuclease-targeting approach CUT&RUN (Skene & Henikoff, eLife 2017) to map R-loops in vivo. We show that this approach accurately identifies R-loops that are sensitive to both Actinomycin D and RNase H and largely overlap with previously identified regions containing R-loops. We also show that MapR can identify R-loops genome-wide from much smaller input material than existing techniques. Overall design: DNA fragments from R-loop were relased by incubating nuclei with GST-RNAseH?-Mnase in vitro (MapR), where ? indicates a mutation that kills the catalytic activity of RNase H. As a control, we incubated the nuclei with GST-Mnase lacking the RNase H moiety (MapR control). We also compared these results with those obtained by expressing a catalytically inactive RNase H fused to the FLAG epitope tag directly in cells and then performing a more conventional CUT&RUN assay (Skene & Henikoff, eLife 2017) using an anti-FLAG antibody. In that case the control is performing the CUT&RUN assay with a non-specific IgG control.