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Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell


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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Figure 1
Figure 2: Examples of use of microscopy with single-cell Hi-C.
Figure 3: FACS setup to sort haploid nuclei.
Figure 4: Agilent Bioanalyzer electropherograms of various Hi-C libraries.
Figure 5: Evaluation of NucProcess reports.
Figure 6: Genome-wide single-cell Hi-C contact maps from several mouse haploid ES cell data sets.
Figure 7: Schematic diagrams of the MoFlo instrument illustrating the arrangement used for FACS sorting of haploid ES cells.
Figure 8: Custom-built microscope for diffraction-limited and superresolution imaging of individual nuclei in 384-well plates.
Figure 9: Determining ploidy and cell-cycle stage of single-cell Hi-C libraries.

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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).

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Authors and Affiliations



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.

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Correspondence to Ernest D Laue.

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Lando, D., Basu, S., Stevens, T. et al. Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell. Nat Protoc 13, 1034–1061 (2018).

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