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Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans

Abstract

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 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.

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Figure 1
Figure 2
Figure 3: Optimization of the crosslinking time.
Figure 4: Optimization of the input amount needed for chromatin immunoprecipitation (ChIP).
Figure 5: Western blot to determine the quality of the polyclonal Ab against DAF-16.
Figure 6: Optimization of the Ab amount for efficient chromatin immunoprecipitation (ChIP).
Figure 7

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Acknowledgements

H.A.T. is a William Randolph Hearst Young Investigator. This project was funded in part by a Burroughs Wellcome Career Award in the Biomedical Sciences to H.A.T., an endowment from the William Randolph Hearst Foundation, and grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK068429 to A.J.M.W.) and National Institute of Aging (AG25891 to H.A.T.).

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A.M. and B.D. contributed equally to the work.

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Correspondence to Heidi A Tissenbaum.

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Mukhopadhyay, A., Deplancke, B., Walhout, A. et al. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3, 698–709 (2008). https://doi.org/10.1038/nprot.2008.38

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