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Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell

Nature Protocols volume 10, pages 1986–2003 (2015)Cite this article

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Abstract

Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C–ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

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Figure 1: Overview of the single-cell Hi-C protocol.
Figure 2: A drawn Pasteur pipette and aspirator tube assembly.
Figure 3: Example image of the cell suspension after Hi-C ligation.
Figure 4: Analysis of Hi-C ligation junctions.
Figure 5: Example gel image of single-cell Hi-C library smears.
Figure 6: Example electropherograms of single-cell Hi-C libraries generated by the Agilent 2100 Bioanalyzer system.
Figure 7: Classification and total read-pair numbers for ten single-cell data sets.
Figure 8: Genome-wide heatmaps created from Hi-C data sets with various quality.

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Acknowledgements

The authors thank I. Clay, N. Cope and K. Tabbada for help and support. This work was supported by the Biotechnology and Biological Sciences Research Council, UK.

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Authors and Affiliations

  1. Nuclear Dynamics Programme, The Babraham Institute, Cambridge, UK

    Takashi Nagano, Steven W Wingett & Peter Fraser

  2. Department of Computer Science and Applied Mathematics and Department of Biological Regulation, Weizmann Institute, Rehovot, Israel

    Yaniv Lubling, Eitan Yaffe & Amos Tanay

  3. Epigenetics Programme, The Babraham Institute, Cambridge, UK

    Wendy Dean

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  1. Takashi Nagano
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  2. Yaniv Lubling
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  4. Steven W Wingett
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  7. Peter Fraser
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Contributions

T.N. and P.F. developed and optimized the protocol. Y.L., E.Y. and A.T. contributed to protocol optimization by developing the processing pipeline for single-cell Hi-C and analyzing the sequence data. S.W.W. contributed to optimize the pipeline. W.D. contributed the single-cell isolation procedure. T.N., S.W.W. and P.F. wrote the manuscript, with inputs from all other authors.

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Correspondence to Takashi Nagano or Peter Fraser.

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Nagano, T., Lubling, Y., Yaffe, E. et al. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell. Nat Protoc 10, 1986–2003 (2015). https://doi.org/10.1038/nprot.2015.127

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