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Chromatin immunoprecipitation and microarray-based analysis of protein location

Abstract

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.

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Figure 1: A sample timeline for the ChIP-Chip protocol.
Figure 2: Results of varying degrees of sonication on fragment size.
Figure 3: 2% agarose gel showing an example of input DNA, DNA after LMPCR amplification and labeled DNA.
Figure 4: Samples of hybridized arrays and scatterplots.
Figure 5: Examples of data from an array that are processed to identify genomic regions that are enriched for binding and verification by gene-specific PCR.

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Acknowledgements

The protocol and suggestions presented here represent the experience accumulated over several years of experimentation by a number of researchers in the lab. In particular, we would like to acknowledge Dmitry Pokholok and Brett Chevalier for their contributions to streamlining the protocol. Other particularly useful contributions or observations were offered by Duncan Odom, Beth Jacobsen, Richard Jenner, Laurie Boyer, Matthew Guenther, Roshan Kumar, Heather Plasterer, David Reynolds and Jane Yoo. We thank current members of the lab, particularly Megan Cole, Matthew Guenther, Beth Jacobsen, Richard Jenner and Michael Kagey, for critical reading of this manuscript.

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Correspondence to Richard A Young.

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T.I.L. and R.A.Y. consult for Agilent Technologies.

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Lee, T., Johnstone, S. & Young, R. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1, 729–748 (2006). https://doi.org/10.1038/nprot.2006.98

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