Genome-Wide Mapping of R-Loops in S. cerevisiae by H-CRAC

R-loops are noncanonical nucleic acid structures formed by an RNA:DNA heteroduplex, presumably arising by the pairing of the nascent RNA to its own DNA template and the subsequent extrusion of a single DNA strand. Such structures, previously thought to be rare and mostly by-products of transcription, are today recognized as physiological intermediates of several molecular processes and to form at much higher rates than anticipated. R-loop levels must be meticulously controlled, as they can lead to DNA damage and genome instability. RNA remodeling, in particular, constitutes a central mechanism for preventing and resolving R-loops. In this chapter, we describe H-CRAC, a procedure to chart R-loops genome-wide, in a strand-specific and high-resolution manner. H-CRAC relies on the capture and sequencing of RNAs UV-crosslinked to tagged RNases H, enzymes that selectively bind and digest RNA:DNA hybrids. In vivo genome-wide profiling of R-loops by H-CRAC reveals unprecedented sensitivity and resolution.