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Determining chromatin architecture with Micro Capture-C

Nature Protocols volume 18, pages 1687–1711 (2023)Cite this article

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Abstract

Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer–promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.

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Fig. 1: Comparison of MCC with other techniques at the Klf1 locus.
Fig. 2: Overview of experimental workflow.
Fig. 3: Data analysis.
Fig. 4: Expected results.

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Data availability

Sequencing data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GSE144336).

Code availability

The code required for analysis of MCC data are available for academic use through the Oxford University Innovation software store (https://process.innovation.ox.ac.uk/software/p/16529a/micro-capture-c-academic/1). A pipeline to run this code is available on GitHub (https://github.com/jojdavies/Micro-Capture-C). Instructions for setting up and running the pipeline are available on github.

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Acknowledgements

J.O.J.D. was supported by grants from Wellcome (098931/Z/12/Z and 225220/Z/22/Z); the MRC (MR/R008108/1 and MC_UU_00029/04); the NIHR Blood and Transplant Research Unit (NIHR203339) and the Oxford Biomedical Research Centre Genomic Medicine and Cell and Gene Therapy Themes (NIHR203311). J.C.H. is supported by an MRC studentship and N.D. was supported by an NIHR Academic Clinical Fellowship (ACF-2019-13-013).

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Author notes

  1. These authors contributed equally: Joseph C. Hamley, Hangpeng Li.

Authors and Affiliations

  1. MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Joseph C. Hamley, Hangpeng Li, Nicholas Denny, Damien Downes & James O. J. Davies

  2. Oxford Biomedical Research Centre, Genomic Medicine and Cell and Gene Therapy Themes, Oxford, UK

    James O. J. Davies

  3. National Institute of Health Research Blood and Transplant Research Unit, Oxford, UK

    James O. J. Davies

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Contributions

J.O.J.D. developed the method and wrote the first draft of the manuscript. J.C.H., H.L. and N.D. optimized the method and wrote the manuscript. D.D. optimized the capture methodology and contributed to the manuscript.

Corresponding author

Correspondence to James O. J. Davies.

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Competing interests

J.O.J.D. is a co-founder of Nucleome Therapeutics and he provides consultancy to the company. He also holds a patent for the MCC method, which is licensed to the company.

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Nature Protocols thanks Argyris Papantonis and Vijay Ramani for their contribution to the peer review of this work.

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Related links

Key papers using this protocol

Hua, P. et al. Nature 595, 125–129 (2021): https://doi.org/10.1038/s41586-021-03639-4

Aljahani, A. et al. Nat. Commun. 13, 2139 (2022): https://doi.org/10.1038/s41467-022-29696-5

Downes, D. J. et al. Nat. Genet. 53, 1606–1615 (2021): https://doi.org/10.1038/s41588-021-00955-3

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Hamley, J.C., Li, H., Denny, N. et al. Determining chromatin architecture with Micro Capture-C. Nat Protoc 18, 1687–1711 (2023). https://doi.org/10.1038/s41596-023-00817-8

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