Abstract
Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.
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Acknowledgements
This work was supported by the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and by a long-term EMBO fellowship to D.U.
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T.S., S.K. and P.F. designed the experiments, T.S. and S.K. developed the method, J.A.M. and D.U. optimized the chromatin immunoprecipitation steps, T.N. developed primers for restriction digestion efficiency tests and T.S. and P.F. wrote the manuscript.
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Supplementary information
Supplementary Fig. 1
Typical raw e4C and ChIP-e4C results, using Hbb-b1 sequence as bait. (PDF 360 kb)
Supplementary Table 1
List of primers used for assessing BglII restriction digestion efficiency in mouse tissues. (PDF 72 kb)
Supplementary Table 2
List of 3C primers used in Anticipated Results section. (PDF 52 kb)
Supplementary Table 3
List of ChIP primers used in Anticipated Results section. (PDF 52 kb)
Supplementary Table 4
List of primers used for assessing e4C bait enrichment in Anticipated Results section. (PDF 51 kb)
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Sexton, T., Kurukuti, S., Mitchell, J. et al. Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip. Nat Protoc 7, 1335–1350 (2012). https://doi.org/10.1038/nprot.2012.071
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DOI: https://doi.org/10.1038/nprot.2012.071
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