Characterization of r-loop structures using single-molecule r-loop footprinting and sequencing

Maika Malig, Frederic Chedin

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages209-228
Number of pages20
DOIs
StatePublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2161
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • DNA:RNA hybrid
  • Non-denaturing bisulfite conversion
  • R-loop
  • SMRT sequencing
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Malig, M., & Chedin, F. (2020). Characterization of r-loop structures using single-molecule r-loop footprinting and sequencing. In Methods in Molecular Biology (pp. 209-228). (Methods in Molecular Biology; Vol. 2161). Humana Press Inc.. https://doi.org/10.1007/978-1-0716-0680-3_15