Abstract

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000–100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

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Acknowledgements

We thank the Cancer Research UK (CRUK) Cambridge Institute for DNA sequencing. This work was supported by the Wellcome Trust (206291/Z/17/Z and 082010/Z/07/Z), the EC FP7 4DCellFate project (277899) and the MRC (MR/P019471/1, MR/M010082/1).

Author information

Author notes

    • Martin Leeb

    Present address: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna, Austria.

    • David Lando
    • , Srinjan Basu
    •  & Tim J Stevens

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, University of Cambridge, Cambridge, UK.

    • David Lando
    • , Srinjan Basu
    • , Kai J Wohlfahrt
    • , Yang Cao
    • , Wayne Boucher
    • , Liam P Atkinson
    •  & Ernest D Laue
  2. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.

    • Tim J Stevens
  3. Wellcome Trust – MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.

    • Andy Riddell
    • , Martin Leeb
    •  & Brian Hendrich
  4. Department of Chemistry, University of Cambridge, Cambridge, UK.

    • Steven F Lee
    •  & Dave Klenerman

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Contributions

D.L. and S.B. developed the experimental part of the protocol with assistance from Y.C. A.R. developed the flow cytometry methods to sort haploid-cell nuclei. T.J.S. and W.B. wrote the data-processing pipeline with assistance from L.P.A. and K.J.W. S.F.L. and D.K. helped to implement microscopy into the protocol. M.L. and B.H. provided expertise in haploid embryonic stem cell culture. E.D.L. supervised the project. D.L., T.J.S. and E.D.L. wrote the manuscript with contributions from all the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ernest D Laue.

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DOI

https://doi.org/10.1038/nprot.2018.017

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