Genome-Wide Native R-Loop Profiling by R-Loop Cleavage Under Targets and Tagmentation (R-Loop CUT&Tag)

Abstract

R-loops are three-stranded nucleic acid structures that consist of a DNA-RNA hybrid and a displaced single-stranded DNA. R-loops occur during transcription and participate in multiple physiological processes such as DNA repair, modulating DNA topology, and regulation of gene transcription. Dysfunctional R-loops associate with several human diseases such as neurological disorders and cancer. Therefore, accurately and comprehensively profiling native R-loops is crucial to understand their functions under both physiological and pathological conditions. Here, we describe a convenient native R-loop profiling method, R-loop CUT&Tag, which combines a DNA-RNA hybrid sensor (GST-His₆-2 × HBD or S9.6 antibody) with a pA-Tn5-based cleavage under targets and tagmentation approach. R-loop CUT&Tag starts with 0.5 million cells and can sensitively detect native and specific R-loops at the promoter, gene body, and enhancer regions.

Keywords: CUT&Tag; DNA–RNA hybrid; Hybrid-binding domain (HBD); R-loop; pA-Tn5.