Quantitative, Convenient, and Efficient Genome-Wide R-Loop Profiling by ssDRIP-Seq in Multiple Organisms

Abstract

R-loop is a three-stranded chromatin structure, comprising one single-stranded DNA and another DNA:RNA hybrid strand, plays various and essential biological functions in many organisms. Developing a precise, efficient, faithful, and unbiased genome-wide R-loop detection method with extensive adaptability in all organisms is at the top priority for R-loop biology. Here, we provide a straightforward and highly efficient protocol for genome-wide strand-specific R-loop profiling in various organisms. In brief, genomic DNA is extracted and fragmented by the cocktail of restriction enzymes, and then the DNA:RNA hybrids are immunoprecipitated, following by the single-stranded DNA adaptor ligation and next-generation sequencing (named as ssDRIP-seq). Coupling with a straightforward and step-by-step bioinformatic pipeline, this method can provide high resolution and comprehensive strand-specific information for R-loop formation. ssDRIP-seq has been successfully applied for detecting R-loops from prokaryotes such as E. coli, to eukaryotes such as S. cerevisiae, mammalian cell culture and tissues, as well as plants Arabidopsis and rice, with high reproducibility and sensitivity.

Keywords: Genome; Next-generation sequencing; Noncoding RNA; Nucleic acid; R-loop.