Immunoprecipitation of RNA: DNA hybrids from budding yeast

Aziz El Hage; David Tollervey

doi:10.1007/978-1-4939-7459-7_8

Immunoprecipitation of RNA: DNA hybrids from budding yeast

Aziz El Hage, David Tollervey

School of Biological Sciences

Research output: Chapter in Book/Report/Conference proceeding › Chapter

Abstract / Description of output

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 language	English
Title of host publication	DNA topoisomerases
Editors	Marc Drolet
Place of Publication	USA
Publisher	Springer Nature
Pages	109-129
Number of pages	21
Volume	1703
Edition	1st
ISBN (Electronic)	978149397459-7
ISBN (Print)	9781493974580
DOIs	https://doi.org/10.1007/978-1-4939-7459-7_8
Publication status	Published - 2018

Publication series

Name	Methods in Molecular Biology
Publisher	Springer
Volume	1703
ISSN (Print)	1064-3745
ISSN (Electronic)	1064-3745

Keywords / Materials (for Non-textual outputs)

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

Access to Document

10.1007/978-1-4939-7459-7_8

Kinetic changes in RNA surveillance and non-coding RNA function during nutrition downshift
Tollervey, D.
UK-based charities
1/10/11 → 31/03/17
Project: Research
Core funding renewal for the Wellcome Trust Centre for Cell Biology
Tollervey, D. & Earnshaw, B.
UK-based charities
1/10/11 → 30/04/17
Project: Research

Cite this

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title = "Immunoprecipitation of RNA: DNA hybrids from budding yeast",

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.",

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

author = "{El Hage}, Aziz and David Tollervey",

note = "Wellcome do not class this a book chapter, but as a journal as it has been indexed in PubMed Central as journal. Even though Methods in Molecular Biology doesn't require OA, Wellcome have, however, agreed to authorise OA. This was subject to Theo's approval to apply for OA Funds. Invoice to be sent to scholcomms@ed.ac.uk 17/04/2018 EN ",

year = "2018",

doi = "10.1007/978-1-4939-7459-7_8",

language = "English",

isbn = "9781493974580",

volume = "1703",

series = "Methods in Molecular Biology",

publisher = "Springer Nature",

pages = "109--129",

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edition = "1st",

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TY - CHAP

T1 - Immunoprecipitation of RNA

T2 - DNA hybrids from budding yeast

AU - El Hage, Aziz

AU - Tollervey, David

N1 - Wellcome do not class this a book chapter, but as a journal as it has been indexed in PubMed Central as journal. Even though Methods in Molecular Biology doesn't require OA, Wellcome have, however, agreed to authorise OA. This was subject to Theo's approval to apply for OA Funds. Invoice to be sent to scholcomms@ed.ac.uk 17/04/2018 EN

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Saccharomyces cerevisiae

KW - RNA:DNA hybrids

KW - R-loop

KW - Chromatin immunoprecipitation (ChIP)

KW - DNA:RNA immunoprecipitation (DRIP)

KW - S9.6 antibody

U2 - 10.1007/978-1-4939-7459-7_8

DO - 10.1007/978-1-4939-7459-7_8

M3 - Chapter

SN - 9781493974580

VL - 1703

T3 - Methods in Molecular Biology

SP - 109

EP - 129

BT - DNA topoisomerases

A2 - Drolet, Marc

PB - Springer Nature

CY - USA

ER -

Immunoprecipitation of RNA: DNA hybrids from budding yeast

Abstract / Description of output

Publication series

Keywords / Materials (for Non-textual outputs)

Access to Document

Fingerprint

Projects

Kinetic changes in RNA surveillance and non-coding RNA function during nutrition downshift

Core funding renewal for the Wellcome Trust Centre for Cell Biology

Cite this