Abstract
R-loops, three-stranded structures containing double-stranded DNA invaded by single-stranded RNA, have been linked to diverse biological processes. They play important roles in regulating gene regulation and DNA repair, contributing to a wide range of diseases. Understanding the formation and dynamic regulation of R-loops is thus a gateway to address many fundamental questions in regulatory biology, which requires the elucidation of the R-loop landscape at the genome scale. To aid in such efforts, this article provides an overview on R-loop mapping strategies along with a detailed protocol based on the use of catalytically inactive RNaseH1, an evolutionarily conserved protein responsible for R-loop recognition and resolution.