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.
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Acknowledgments
We thank Stephan Hamperl and Joshua Brickner for valuable feedback. This work was supported by the Leukemia and Lymphoma Society [5455-17 to M.P.C.]; National Institutes of Health [GM119334 to K.A.C., S10OD018220 to the Stanford Functional Genomics Facility]. K.A.C. is an ACS Research Professor.
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Crossley, M.P., Cimprich, K.A. (2022). Quantitative DNA–RNA Immunoprecipitation Sequencing with Spike-Ins. In: Aguilera, A., Ruzov, A. (eds) R-Loops . Methods in Molecular Biology, vol 2528. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2477-7_26
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DOI: https://doi.org/10.1007/978-1-0716-2477-7_26
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