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.
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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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El Hage, A., Tollervey, D. (2018). Immunoprecipitation of RNA:DNA Hybrids from Budding Yeast. In: Drolet, M. (eds) DNA Topoisomerases. Methods in Molecular Biology, vol 1703. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7459-7_8
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DOI: https://doi.org/10.1007/978-1-4939-7459-7_8
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