Detection of R-Loop Structures by Immunofluorescence Using the S9.6 Monoclonal Antibody

This protocol describes a method of detection of R-loop structures by Immunofluorescence using the S9.6 antibody. R-loops are three-stranded nucleic acid structures that comprise the nascent RNA hybridized with the DNA template strand (RNA–DNA hybrid) leaving the nontemplate DNA strand single-stranded (ssDNA). R-loops are dynamic structures that have been linked to transcription-associated DNA damage and genomic instability in certain contexts but they also possess critical regulatory functions. They are direct products of transcription and they have been associated with transcriptional activation, repression and termination in cases of both protein-coding and noncoding genes. Visualizing and mapping R-loops has been a sought-after task over the last years. Next-generation sequencing of RNA–DNA hybrids, which are components of R-loops, using the S9.6 antibody, aims to detect R-loops genome-wide, whereas Immunofluorescence is performed to visualize R-loops in single cells. While mapping R-loops genome-wide is very important for identifying and studying their location-specific role, microscopy offers the advantage of spatial information and the ability to quantify them on a single cell level. In this chapter, I will describe the protocol I have used to image RNA–DNA hybrids in the nucleus of mammalian cells.