Genome-wide profiling of R-loop by affinity-independent and nuclease-based method [RIAN-seq]

R-loop is abundant triplex nucleic strand in organism, but many physiological and pathological functions remain to be fully understood, owing to a lack of robust and high-resolution methods for genome-wide detection. In this study, we develop R-loop identification assisted by nuclease and high-throughput sequencing (RIAN-seq), a method to identify and quantify R-loop stoichiometry at affinity-independent enrichment. We reveal that RIAN-seq has ultrahigh sensitivity, accuracy and reliability for identifying R-loop sites, compared with all existing methods. In addition, RIAN-seq enables capturing previously unidentified clustered R-loop sites with distinct patterns. In sum, we reveal that RIAN-seq can not only quantitatively dissect the R-loop landscape at unprecedented resolution, but also provide an unbiased and convenient method for R-loop biology.