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Characterization of R-Loop Structures Using Single-Molecule R-Loop Footprinting and Sequencing

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RNA-Chromatin Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2161))

Abstract

R-loops are three-stranded structures that form during transcription when the nascent RNA hybridizes with the template DNA resulting in a DNA:RNA hybrid and a looped-out single-stranded DNA (ssDNA) strand. These structures are important for normal cellular processes and aberrant R-loop formation has been implicated in a number of pathological outcomes, including certain cancers and neurodegenerative diseases. Mapping R-loops has primarily been performed using DRIP (DNA:RNA immunoprecipitation) based methods that are dependent on the anti-DNA:RNA hybrid S9.6 antibody and short-read sequencing. While DRIP-based methods are robust and report R-loop formation genome-wide, they only do so at the population average level; interrogating R-loop formation at the single molecule level is not feasible with such approaches. Here we present single molecule R-loop footprinting (SMRF-seq), a method that relies on the chemical reactivity of the displaced ssDNA strand to non-denaturing sodium bisulfite and single molecule long-read sequencing as a readout, to characterize R-loops. SMRF-seq can be used independently of S9.6 to generate high resolution, strand-specific, maps of individual R-loops at ultra-deep coverage on kilobases-length DNA fragments.

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Acknowledgements

We thank Chedin lab members for useful discussions, Dr. Lionel A. Sanz for constructive comments on the manuscript, and Dr. Stella R. Hartono for developing the Gargamel analysis pipeline. This work was funded by the National Institutes of Health (Grant R01 GM120607 to F.C.) and was supported, in part, by National Science Foundation Graduate Research Fellowship (Grant 1650042 to M.M.) and National Institute of General Medical Sciences Biomolecular Technology Predoctoral T32 Training Program (Grant T32-GM008799 to M.M.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology and Genome Center, University of California Davis, Davis, CA, USA

    Maika Malig & Frederic Chedin

  2. Integrative Genetics and Genomics Graduate Group, University of California Davis, Davis, CA, USA

    Maika Malig

Authors
  1. Maika Malig
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  2. Frederic Chedin
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Correspondence to Frederic Chedin .

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Editors and Affiliations

  1. Department for Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark

    Ulf Andersson Vang Ørom

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Malig, M., Chedin, F. (2020). Characterization of R-Loop Structures Using Single-Molecule R-Loop Footprinting and Sequencing. In: Ørom, U. (eds) RNA-Chromatin Interactions. Methods in Molecular Biology, vol 2161. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0680-3_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0680-3_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0679-7

  • Online ISBN: 978-1-0716-0680-3

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