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qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide
R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to m...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641308/ https://www.ncbi.nlm.nih.gov/pubmed/32544226 http://dx.doi.org/10.1093/nar/gkaa500 |
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author | Crossley, Magdalena P Bocek, Michael J Hamperl, Stephan Swigut, Tomek Cimprich, Karlene A |
author_facet | Crossley, Magdalena P Bocek, Michael J Hamperl, Stephan Swigut, Tomek Cimprich, Karlene A |
author_sort | Crossley, Magdalena P |
collection | PubMed |
description | R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA–DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA–RNA immunoprecipitation (qDRIP), a method combining synthetic RNA–DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA–DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA–DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights. |
format | Online Article Text |
id | pubmed-7641308 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-76413082020-11-10 qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide Crossley, Magdalena P Bocek, Michael J Hamperl, Stephan Swigut, Tomek Cimprich, Karlene A Nucleic Acids Res Methods Online R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA–DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA–RNA immunoprecipitation (qDRIP), a method combining synthetic RNA–DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA–DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA–DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights. Oxford University Press 2020-06-16 /pmc/articles/PMC7641308/ /pubmed/32544226 http://dx.doi.org/10.1093/nar/gkaa500 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Crossley, Magdalena P Bocek, Michael J Hamperl, Stephan Swigut, Tomek Cimprich, Karlene A qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title | qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title_full | qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title_fullStr | qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title_full_unstemmed | qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title_short | qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide |
title_sort | qdrip: a method to quantitatively assess rna–dna hybrid formation genome-wide |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641308/ https://www.ncbi.nlm.nih.gov/pubmed/32544226 http://dx.doi.org/10.1093/nar/gkaa500 |
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