Immunoprecipitation of RNA: DNA hybrids from budding yeast

Research output: Chapter in Book/Report/Conference proceedingChapter

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.
Original languageEnglish
Title of host publicationDNA topoisomerases
EditorsMarc Drolet
Place of PublicationUSA
PublisherSpringer Nature
Pages109-129
Number of pages21
Volume1703
Edition1st
ISBN (Electronic)978149397459-7
ISBN (Print)9781493974580
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in Molecular Biology
PublisherSpringer
Volume1703
ISSN (Print)1064-3745
ISSN (Electronic)1064-3745

Keywords

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

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