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ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos

Nature Protocols volume 1, pages 2839–2855 (2006)Cite this article

Abstract

This protocol describes a method to detect in vivo associations between proteins and DNA in developing Drosophila embryos. It combines formaldehyde crosslinking and immunoprecipitation of protein-bound sequences with genome-wide analysis using microarrays. After crosslinking, nuclei are enriched using differential centrifugation and the chromatin is sheared by sonication. Antibodies specifically recognizing wild-type protein or, alternatively, a genetically encoded epitope tag are used to enrich for specifically bound DNA sequences. After purification and polymerase chain reaction-based amplification, the samples are fluorescently labeled and hybridized to genomic tiling microarrays. This protocol has been successfully used to study different tissue-specific transcription factors, and is generally applicable to in vivo analysis of any DNA-binding proteins in Drosophila embryos. The full protocol, including the collection of embryos and the collection of raw microarray data, can be completed within 10 days.

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Figure 1: Schematical overview of ChIP-on-chip experiments.
Figure 2: Overview of the steps outlined in this protocol.
Figure 3: Enriched sequences can be identified by comparing ChIPs performed with specific anti-Mef2 or preimmune antisera.
Figure 4: Crosslinked chromatin can be sheared reproducibly by sonication.
Figure 5: Enrichment of a known Mef2-binding site within the Act57B locus.
Figure 6: SAM analysis using TM4's MeV module reveals sequences significantly enriched in anti-Mef2 but not in mock ChIPs.

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Acknowledgements

We are grateful to the laboratories of Robert White, Steve Russell (Cambridge) and Renato Paro (Basel) for sharing their ChIP (on chip) protocols with us.

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  1. EMBL Heidelberg, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Thomas Sandmann, Janus S Jakobsen & Eileen E M Furlong

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  1. Thomas Sandmann
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  3. Eileen E M Furlong
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Correspondence to Eileen E M Furlong.

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Sandmann, T., Jakobsen, J. & Furlong, E. ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos. Nat Protoc 1, 2839–2855 (2006). https://doi.org/10.1038/nprot.2006.383

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