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Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP

Abstract

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type–specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type–specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.

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Figure 1: Schematic representation of the BiTS–ChIP-seq protocol.
Figure 2: Mesoderm-specific nuclei are stained, extracted and sorted to purity.
Figure 3: Cell sorter configuration used in the acquisition and sorting of mesodermal D. melanogaster nuclei.
Figure 4: Representative flow cytometric data, regions and sort gate.
Figure 5: Solexa sequencing library preparation from tissue-specific, sheared chromatin.

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Acknowledgements

We thank E.H. Gustafson for fly work, and J. Erceg for sharing staged, sorted nuclei. We are very grateful to all members of the Furlong Laboratory, J. Mueller, C. Margulies and A.G. Ladurner for helpful discussions. This work was supported by grants to E.E.M.F. from ERASysBio (Mod Heart) and the Human Frontiers Science Organization, and by a long-term fellowship to R.P.Z. from the International Human Frontiers Science Program Organization. S.B. was funded by the European Molecular Biology Laboratory Interdisciplinary Postdoctoral Programme.

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S.B., R.P.Z. and E.E.M.F. designed the study. A.P.-G. and A.R. conducted the FACS experiments. S.B. and R.P.Z. conducted all other experiments. S.B., R.P.Z., A.P.-G., A.R., A.-C.G. and E.E.M.F. wrote the manuscript.

Corresponding author

Correspondence to Eileen E M Furlong.

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The authors declare no competing financial interests.

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Bonn, S., Zinzen, R., Perez-Gonzalez, A. et al. Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP. Nat Protoc 7, 978–994 (2012). https://doi.org/10.1038/nprot.2012.049

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  • DOI: https://doi.org/10.1038/nprot.2012.049

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