Abstract
R-loops are three-stranded, DNA:RNA hybrid-containing structures that form naturally throughout the genome as a consequence of transcription. Accurately determining the genomic locations and strand of origin of R-loops is critical to understanding their roles in gene regulation and disease. Here, we describe a nuclease-based protocol for genome-wide and strand-specific R-loop detection, which we term MapR. This method targets native R-loops for cleavage and release using a modified RNase H enzyme, followed by deep sequencing. An extension of the protocol, BisMapR, can additionally introduce strand specificity via non-denaturing bisulfite conversion of the R-loop's single-stranded DNA component. MapR and BisMapR identify R-loops with high resolution and low background, can be performed with low cell input, and require short experimental time.
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