Abstract
R-loops are abundant, RNA-containing chromatin structures that form in the genomes of both eukaryotes and prokaryotes. Devising methods to identify the precise genomic locations of R-loops is critical to understand how these structures regulate numerous cellular processes, including replication, termination, and chromosome segregation, and how their unscheduled formation results in disease. Here, we describe a new, highly sensitive, and antibody-independent method, MapR, to profile native R-loops genome wide. MapR takes advantage of the natural specificity of the RNase H enzyme to recognize DNA:RNA hybrids, a defining feature of R-loops, and combines it with a CUT&RUN approach to target, cleave, and release R-loops that can then be sequenced. MapR has low background, is faster than current R-loop detection technologies, and can be performed in any cell type without the need to generate stable cell lines. © 2020 by John Wiley & Sons, Inc.