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Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins.

ABSTRACT: R-loops are three-stranded nucleic acid structures, comprising an RNA-DNA hybrid and a displaced strand of ssDNA. R-loops have important physiological roles in cells, but deregulation of R-loop dynamics can also have harmful cellular outcomes. The genome-wide mapping of R-loops offers an unbiased approach to study R-loop biology in a wide range of contexts. Here we present a protocol to sequence RNA-DNA hybrids genome-wide with strand-specificity and high resolution. We also include information on how to prepare and incorporate into the workflow appropriate internal spike-in standards which facilitate accurate normalization of the sequencing signal, thereby providing quantitative insights into R-loop formation between different experimental samples.

SUBMITTER: Crossley MP

PROVIDER: S-EPMC9942688 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins.

Crossley Magdalena P MP Cimprich Karlene A KA

Methods in molecular biology (Clifton, N.J.) 20220101

R-loops are three-stranded nucleic acid structures, comprising an RNA-DNA hybrid and a displaced strand of ssDNA. R-loops have important physiological roles in cells, but deregulation of R-loop dynamics can also have harmful cellular outcomes. The genome-wide mapping of R-loops offers an unbiased approach to study R-loop biology in a wide range of contexts. Here we present a protocol to sequence RNA-DNA hybrids genome-wide with strand-specificity and high resolution. We also include information ...[more]

PMID: 35704206

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