Detection and characterization of R loop structures

R loops are special three stranded nucleic acid structures that comprise a nascent RNA hybridized with the DNA template strand, leaving a non-template DNA single-stranded. More specifically, R loops form in vivo as G-rich RNA transcripts invade the DNA duplex and anneal to the template strand to generate an RNA:DNA hybrid, leaving the non-template, G-rich DNA strand in a largely single-stranded conformation (Aguilera and Garcia-Muse, Mol Cell 46:115–124, 2012). DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation. RNA-DNA hybrids influence genomic instability, and growing evidence points to an important role for R loops in active gene expression regulation (Ginno et al., Mol Cell 45, 814–825, 2012; Sun et al., Science 340: 619–621, 2013; Bhatia et al., Nature 511, 362–365, 2014). Analysis of the occurrence of such structures is therefore of increasing relevance and herein we describe methods for the in vivo and in vitro identification and characterization of R loops in mammalian systems