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High-resolution circular chromosome conformation capture assay

Nature Protocols volume 3, pages 303–313 (2008)Cite this article

Abstract

The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2–3 weeks.

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Figure 1: The principle of the high-resolution 4C method.
Figure 2: High-resolution 4C analysis of the human chromosome 11.
Figure 3: Agarose gel electrophoresis of formaldehyde cross-linked, restriction-enzyme-digested chromatin DNA.
Figure 4: Scheme to assess the efficiency of restriction enzyme digestion by Q-PCR.

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Acknowledgements

We gratefully acknowledge help with the processing of NimbleGen data from Kuljeet Singh and discussions with many members of the RO lab. This work was supported by grants from HEROIC (EU), Swedish Research Council, Swedish Cancer Foundation, Swedish Pediatric Cancer Foundation and Lundberg's Foundation.

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Authors and Affiliations

  1. Department of Development and Genetics, Uppsala University, Norbyvägen 18A, Uppsala, S-752 36, Sweden

    Anita Göndör, Carole Rougier & Rolf Ohlsson

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  1. Anita Göndör
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  2. Carole Rougier
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  3. Rolf Ohlsson
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Correspondence to Anita Göndör.

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Göndör, A., Rougier, C. & Ohlsson, R. High-resolution circular chromosome conformation capture assay. Nat Protoc 3, 303–313 (2008). https://doi.org/10.1038/nprot.2007.540

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