R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1

• Published in: Nature protocols Vol. 14; no. 5; pp. 1661 - 1685
• Main Authors: Chen, Jia-Yu, Zhang, Xuan, Fu, Xiang-Dong, Chen, Liang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.05.2019; Nature Publishing Group
• Subjects: 631/1647/2217/2088; 631/337/572; 631/61/212; Analytical Chemistry; Animals; Biological activity; Biological Techniques; Biomedical and Life Sciences; Cell Line; Cell lines; Chromatin; Chromatin Immunoprecipitation - methods; Chromosome Mapping - methods; Computational Biology/Bioinformatics; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA damage; DNA repair; DNA structure; DNA-directed RNA polymerase; Gene expression; Gene mapping; Genetic aspects; Genetic research; Genomes; Genomic instability; Genomics; Hybrid structures; Hybrids; Hydrolases; Immunoprecipitation; Life Sciences; Mapping; Methods; Microarrays; Monoclonal antibodies; Mutation; Myelodysplastic syndrome; Organic Chemistry; Protocol; R-loops; Ribonuclease H - chemistry; Ribonuclease H - genetics; Ribonuclease H - metabolism; Ribonucleic acid; RNA; RNA - chemistry; RNA - genetics; RNA polymerase; RNA polymerase II; Splicing; Splicing factors; Stability; Structure; Transcription; United States
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-019-0154-6

Nascent RNA may form a three-stranded structure with DNA, called an R-loop, which has been linked to fundamental biological processes such as transcription, replication and genome instability. Here, we provide a detailed protocol for a newly developed strategy, named R-ChIP, for robust capture of R-loops genome-wide. Distinct from R-loop-mapping methods based on the monoclonal antibody S9.6, which recognizes RNA–DNA hybrid structures, R-ChIP involves expression of an exogenous catalytically inactive RNASEH1 in cells to bind RNA–DNA hybrids but not resolve them. This is followed by chromatin immunoprecipitation (ChIP) of the tagged RNASEH1 and construction of a strand-specific library for deep sequencing. It takes ~3 weeks to establish a stable cell line expressing the mutant enzyme and 5 more days to proceed with the R-ChIP protocol. In principle, R-ChIP is applicable to both cell lines and animals, as long as the catalytically inactive RNASEH1 can be expressed to study the dynamics of R-loop formation and resolution, as well as its impact on the functionality of the genome. In our recent studies with R-ChIP, we showed an intimate spatiotemporal relationship between R-loops and RNA polymerase II pausing/pause release, as well as linking augmented R-loop formation to DNA damage response induced by driver mutations of key splicing factors associated with myelodysplastic syndrome (MDS). This protocol describes the steps in R-ChIP, a procedure based on chromatin immunoprecipitation (ChIP) of an exogenously expressed mutant RNASEH1 to capture the genome-wide distribution of R-loops.