Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification

Nucleic Acids Research
Harm van BakelH Th Marc Timmers

Abstract

Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein-DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.

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Citations

Jun 7, 2011·Nature Methods·Pattabhiraman ShankaranarayananHinrich Gronemeyer
Jul 2, 2011·Nature Protocols·Wieteke A M HoeijmakersHendrik G Stunnenberg
Jan 28, 2012·Nature Protocols·Pattabhiraman ShankaranarayananHinrich Gronemeyer
Sep 22, 2009·Nature Structural & Molecular Biology·Folkert J van WervenH Th Marc Timmers
Aug 16, 2008·Genes & Development·Folkert J van WervenH Th Marc Timmers
Apr 20, 2014·PLoS Genetics·Yujia A ChanPhilip Hieter
Nov 22, 2012·Blood·Yaeko Nakajima-TakagiAtsushi Iwama
Aug 19, 2009·FEMS Yeast Research·Bernard TurcotteNitnipa Soontorngun
Jul 16, 2009·Genesis : the Journal of Genetics and Development·Meghan E MinardMichelle Craig Barton
Dec 17, 2016·The EMBO Journal·Wim J de JongeFrank Cp Holstege
Jun 24, 2018·Genome Research·Blanca Gómez-Escoda, Pei-Yun Jenny Wu
Feb 22, 2018·PLoS Genetics·Anthony PerrotPei-Yun Jenny Wu
Sep 21, 2019·Nature Communications·Emily Yun-Chia ChangPeter C Stirling
Nov 17, 2021·The Journal of Cell Biology·Daniel A BartlettDavid M Gilbert
May 21, 2011·Molecular Cell·Tineke L LenstraFrank C P Holstege

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Methods Mentioned

BETA
immunoprecipitation
ChIP
DamID
PCR
ChIP-chip
in vitro transcription
ChIPs

Software Mentioned

ENSEMBL
MAANOVA
GIBBS
Imagene

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