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Chromatin tracing and multiplexed imaging of nucleome architectures (MINA) and RNAs in single mammalian cells and tissue

Nature Protocols volume 16, pages 2667–2697 (2021)Cite this article

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Abstract

The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue and how organization affects gene expression remains a major challenge. Previous approaches have been limited by a lack of capacity to directly trace chromatin folding in 3D and to simultaneously measure genomic organization in relation to other nuclear components and gene expression in the same single cells. We have developed an image-based 3D genomics technique termed ‘chromatin tracing’, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. More recently, we also developed multiplexed imaging of nucleome architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli and copy numbers of numerous RNA species in the same single cells in mammalian tissue. Here, we provide detailed protocols for chromatin tracing in cell lines and MINA in mammalian tissue, which take 3–4 d for experimental work and 2–3 d for data analysis. We expect these developments to be broadly applicable and to affect many lines of research on 3D genomics by depicting multiscale genomic architectures associated with gene expression, in different types of cells and tissue undergoing different biological processes.

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Fig. 1: Schematic diagram of the chromatin-tracing and MINA protocols.
Fig. 2: Primary probe synthesis.
Fig. 3: Outline of the MINA method.
Fig. 4: Outline of MINA sequential hybridization and imaging.
Fig. 5: Data analysis with MinaAnalyst.
Fig. 6: Schematic illustration of the home-built microscope and focus-lock system.
Fig. 7: Example results of single-copy and ensemble analyses of chromatin tracing.
Fig. 8: Example results of gene expression and cell type–specific genome architecture analyses.

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Data availability

An example raw dataset of two imaging fields is downloadable from https://campuspress.yale.edu/wanglab/mina-analyst/. The full datasets used to generate Figs. 7 and 8 are not uploaded online because of the prohibitively large size. The full datasets are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The ProbeDealer package can be downloaded from https://campuspress.yale.edu/wanglab/probedealer. The MinaAnalyst package can be downloaded from https://campuspress.yale.edu/wanglab/mina-analyst/.

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Acknowledgements

S.W. is supported by NIH Director’s New Innovator Award 1DP2GM137414, NCI grant 1R33CA251037, NHGRI grant 1R01HG011245 and 4DN NCI grant 1U01CA260701. This work is partially supported by NHGRI grant 1R01HG011245. M.H. and Y.Cheng are supported by a China Scholarship Council (CSC) Grant. J.S.D.R. is supported by an NIH Predoctoral Training Grant (2T32GM007499). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Miao Liu, Bing Yang, Mengwei Hu, Jonathan S. D. Radda, Yanbo Chen, Shengyan Jin, Yubao Cheng & Siyuan Wang

  2. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Siyuan Wang

  3. Yale Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA

    Siyuan Wang

  4. Molecular Cell Biology, Genetics and Development Program, Yale University, New Haven, CT, USA

    Siyuan Wang

  5. Biochemistry, Quantitative Biology, Biophysics and Structural Biology Program, Yale University, New Haven, CT, USA

    Siyuan Wang

  6. M.D.-Ph.D. Program, Yale University, New Haven, CT, USA

    Siyuan Wang

  7. Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA

    Siyuan Wang

  8. Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA

    Siyuan Wang

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Contributions

S.W. conceived and developed the chromatin-tracing technique with help from others and conceived the MINA technique. M.L. performed experiments in developing MINA with help from others. M.H., B.Y. and S.W. built the ProbeDealer package with help from others. S.W. built the MinaAnalyst package. M.L. and S.W. wrote the manuscript with inputs from B.Y., M.H., J.S.D.R., Y. Chen, S.J. and Y. Cheng.

Corresponding author

Correspondence to Siyuan Wang.

Ethics declarations

Competing interests

S.W. is one of the inventors on a patent applied for by Harvard University related to MERFISH. The other authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Vadim Backman and Srinjan Basu for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Wang, S. et al. Science 353, 598–602 (2016): https://doi.org/10.1126/science.aaf8084

Liu, M. et al. Nat. Commun. 11, 2907 (2020): https://doi.org/10.1038/s41467-020-16732-5

Hu, M. et al. Sci. Rep. 10, 22031 (2020): https://doi.org/10.1038/s41598-020-76439-x

Key data used in this protocol

Liu, M. et al. Nat. Commun. 11, 2907 (2020): https://doi.org/10.1038/s41467-020-16732-5

Supplementary information

Reporting Summary

Supplementary Table 1

Sequences of the forward primers, reverse primers and reverse transcription primers for primary probe synthesis

Supplementary Table 2

Sequences of Alexa Fluor 750–labeled MERFISH secondary probes, Alexa Fluor 647–labeled chromatin-tracing secondary probes and ATTO 565–labeled chromatin-tracing secondary probes

Source data

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8a

Unprocessed images.

Source Data Fig. 8b–e

Statistical source data.

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Liu, M., Yang, B., Hu, M. et al. Chromatin tracing and multiplexed imaging of nucleome architectures (MINA) and RNAs in single mammalian cells and tissue. Nat Protoc 16, 2667–2697 (2021). https://doi.org/10.1038/s41596-021-00518-0

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