spKAS-seq reveals R-loop dynamics using low-input materials by detecting single-stranded DNA with strand specificity

R-loops affect transcription and genome stability. Dysregulation of R-loops is related to human diseases. Genome-wide R-loop mapping typically uses the S9.6 antibody or inactive ribonuclease H, both requiring a large number of cells with varying results observed depending on the approach applied. Here, we present strand-specific kethoxal-assisted single-stranded DNA (ssDNA) sequencing (spKAS-seq) to map R-loops by taking advantage of the presence of a ssDNA in the triplex structure. We show that spKAS-seq detects R-loops and their dynamics at coding sequences, enhancers, and other intergenic regions with as few as 50,000 cells. A joint analysis of R-loops and chromatin-bound RNA binding proteins (RBPs) suggested that R-loops can be RBP binding hotspots on the chromatin.

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All sequencing data generated in this study are available at NCBI Gene Expression Omnibus (GEO) with the accession number: GSE192822. Public data used in this study are summarized in data file S1. Mapping statistics of high-throughput sequencing data generated in this study are provided in data file S2. Custom code used in this study has been deposited to Zenodo with the accession number 6519166 (https://zenodo.org/record/6519166#.Yp93bKjMJu8).