Best practices for the visualization, mapping, and manipulation of R-loops

R-loops represent an abundant class of large non-B DNA structures in genomes. Even though they form transiently and at modest frequencies, interfering with R-loop formation or dissolution significantly impacts genome stability. Addressing the mechanism(s) of R-loop-mediated genome destabilization requires a precise characterization of their distribution in genomes. A number of independent methods have been developed to visualize and map R-loops, but their results are at times discordant, leading to confusion. Here we review the main existing methodologies underlying R-loop mapping and assess their limitations and the robustness of existing datasets. We offer a set of best practices to improve the reproducibility of maps, hoping that such guidelines could be useful for authors and referees alike. Finally, we offer a possible resolution to the apparent contradictions in R-loop mapping outcomes between antibody-based and RNase H1-based mapping approaches. Overall design: DRIPc-seq was performed on human HeLa and K562 cells with and without RNase H treatment. DRIP-seq was performed on human HeLa cells.