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

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-...

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Bibliographic Details
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: England Nature Publishing Group 01.05.2019
Subjects:
Animals
Biological activity
Cell Line
Cell lines
Chromatin
Chromatin Immunoprecipitation - methods
Chromosome Mapping - methods
Deoxyribonucleic acid
DNA
DNA - chemistry
DNA - genetics
DNA damage
DNA structure
DNA-directed RNA polymerase
Gene expression
Gene mapping
Genomes
Genomic instability
Hybrid structures
Hybrids
Immunoprecipitation
Mapping
Monoclonal antibodies
Mutation
Myelodysplastic syndrome
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
Transcription
ISSN:1754-2189, 1750-2799, 1750-2799
Online Access:Get full text
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Summary: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).
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ISSN:1754-2189
1750-2799
1750-2799
DOI:10.1038/s41596-019-0154-6