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

R-loops impact transcription and genome stability and are related to human diseases. Genome-wide R-loop mapping typically uses the S9.6 antibody or inactive RNase H, both requiring a large number of cells with varying results observed depending on the approach applied. Here, we present strand-specific KAS-seq (spKAS-seq) to map R-loops by taking advantage of the presence of a single-stranded DNA (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 and eliminates potential bias towards open chromatin and RNase H-sensitive regions. 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. Identification of genome-wide R-loops using strand-specific KAS-seq in HEK293, HepG2 and K562 cells.