Immunoprecipitation of RNA:DNA Hybrids from Budding Yeast

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

During transcription, the nascent transcript behind an elongating RNA polymerase (RNAP) can invade the DNA duplex and hybridize with the complementary DNA template strand, generating a three-stranded “R-loop” structure, composed of an RNA:DNA duplex and an unpaired non-template DNA strand. R-loops can be strongly associated with actively transcribed loci by all RNAPs including the mitochondrial RNA polymerase (mtRNAP). In this chapter, we describe two protocols for the detection of RNA:DNA hybrids in living budding yeast cells, one that uses conventional chromatin immunoprecipitation (ChIP-qPCR) and one that uses DNA:RNA immunoprecipitation (DRIP-qPCR). Both protocols make use of the S9.6 antibody, which is believed to recognize the intermediate A/B helical RNA:DNA duplex conformation, with no sequence specificity.

Key words

Saccharomyces cerevisiae RNA:DNA hybrids R-loop Chromatin immunoprecipitation (ChIP) DNA:RNA immunoprecipitation (DRIP) S9.6 antibody 

Notes

Acknowledgments

We thank Kim Kotovic for initial help with the ChIP technique, members of Jean Beggs lab for giving us access to the bioruptor PICO, and Shaun Webb for help with bioinformatics analysis. We thank Andres Aguilera, Frederic Chedin, Martin Reijns, and Leonel Sanz for sharing protocols and/or reagents. We thank Frederic Chedin, Benoit Palancade, and Ralf Wellinger for critically reading the manuscript. We apologise to the colleagues whose work is not cited in this chapter due to space constraints. This work was supported by a Wellcome Trust Fellowship to DT (077248) and by core funding to the Wellcome Trust Centre for Cell Biology (092076).

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Wellcome Centre for Cell BiologyUniversity of EdinburghEdinburghUK

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