Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans.
In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes approximately 10-12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations.
Mukhopadhyay
Arnab
A
Program in Gene Function and Expression, Program in Molecular Medicine, Aaron Lazare Research Building, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA.
Deplancke
Bart
B
Walhout
Albertha J M
AJ
Tissenbaum
Heidi A
HA
D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
D014157:Transcription Factors; D004247:DNA