Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins

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 ap...

Full description

Saved in:
Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 2528; p. 381
Main Authors Crossley, Magdalena P, Cimprich, Karlene A
Format Journal Article
LanguageEnglish
Published United States 2022
Subjects
Chromosome Mapping
DNA - chemistry
DNA - genetics
Immunoprecipitation
R-Loop Structures
RNA - chemistry
RNA - genetics
R-Loops
qDRIP (quantitative DNA–RNA immunoprecipitation)
S9.6 antibody
Next-generation sequencing
RNA–DNA hybrids
Spike-in
Online AccessGet more information

Cover

More Information
Summary: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.
ISSN:1940-6029
DOI:10.1007/978-1-0716-2477-7_26