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RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure

Nature Protocols volume 17, pages 1306–1331 (2022)Cite this article

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Abstract

DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the increasing resolution offered by super-resolution microscopy, the optimal maintenance of chromatin structure within the nucleus is essential for accuracy in measurements and interpretation of data. However, standard 3D-FISH requires potentially destructive heat denaturation in the presence of chaotropic agents such as formamide to allow access to the DNA strands for labeled FISH probes. To avoid the need to heat-denature, we developed Resolution After Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease digestion to generate single-stranded target DNA for efficient probe binding over a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of nuclear proteins or the detection of RNAs. Here, we provide detailed procedures for RASER-FISH in mammalian cultured cells to detect single loci, chromatin tracks and topologically associating domains with conventional and super-resolution 3D structured illumination microscopy. Moreover, we provide a validation and characterization of our method, demonstrating excellent preservation of chromatin structure and nuclear integrity, together with improved hybridization efficiency, compared with classic 3D-FISH protocols.

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Fig. 1: Comparison of prehybridization workflow for RASER-FISH with heat denaturation FISH techniques.
Fig. 2: Comparison of hybridization strategies.
Fig. 3: Comparison of chromatin structure and TAD shapes.
Fig. 4: RASER-FISH compared with heat-denaturation FISH.
Fig. 5: Workflow for RASER-FISH.
Fig. 6: RASER-FISH hybridization examples with inset magnifications.

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Data availability

Figures 2, 3 and 6 have associated raw image data plus one dataset. All raw data files are archived in Figshare: Fig. 2 at https://doi.org/10.6084/m9.figshare.16778899, Fig. 3 at https://doi.org/10.6084/m9.figshare.16778902 and Fig. 6 at https://doi.org/10.6084/m9.figshare.16755394. Source data are provided with this paper.

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Acknowledgements

We thank C. Lagerholm for extensive imaging support and D. Higgs for long-term support during the development of this technique. We thank R. Klose, J. and C. Lukas, F. Ochs, D. Higgs and J. Hughes for cells and images prepared during collaborations with them. We thank T. Brown and A. El-Sagheer for development and oversight of the oligonucleotide probe generation, and E. Heard for BAC RP24-217l10. Work in the Buckle laboratory was supported by MRC grants MC_UU_00016/1 and MR/N00969X/1, the latter in collaboration with J. Hughes, and by BBSRC grant BB/L01811X held in collaboration with T. Brown (Department of Physical Chemistry, Oxford University) and further supported by the Wolfson Imaging Centre Oxford funded by the Wolfson Foundation 18272, joint MRC/BBSRC/EPSRC MR/K015777X/1, Wellcome Trust Multi-User Equipment 104924/Z/14/Z. 3D-SIM imaging was performed at the Micron Oxford Advanced Bioimaging Unit funded by a Wellcome Trust Strategic Award 091911 and 107457/Z/15/Z. L.S. further acknowledges support by the EU Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement no. 766181. E.P. was sponsored by the International Internship Program (IIP) at Princeton University.

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  1. Eva Parisi

    Present address: School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, UK

Authors and Affiliations

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

    Jill M. Brown, Sara De Ornellas, Eva Parisi & Veronica J. Buckle

  2. Micron Oxford Advanced Bioimaging Unit, Department of Biochemistry, Oxford University, Oxford, UK

    Lothar Schermelleh

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Contributions

J.M.B. and V.J.B. developed the protocol and hybridized, imaged and analyzed RASER-FISH preparations, S.D.O. synthesized, hybridized and analyzed the oligonucleotide probe preparations, L.S. and E.P. hybridized, imaged and analyzed preparations using structured illumination miroscopy, and J.M.B., L.S. and V.J.B. wrote the paper.

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Correspondence to Veronica J. Buckle.

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Nature Protocols thanks Marion Cremer, Michael Hausmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Brown, J. et al. Nat. Commun. 9, 3849 (2018): https://doi.org/10.1038/s41467-018-06248-4

Ochs, F. et al. Nature 574, 571–574 (2019): https://doi.org/10.1038/s41586-019-1659-4

Miron, E. et al. Sci. Adv. 6, eaba8811 (2020): https://doi.org/10.1126/sciadv.aba8811

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Supplementary Methods 1 and 2 and Supplementary References.

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Brown, J.M., De Ornellas, S., Parisi, E. et al. RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protoc 17, 1306–1331 (2022). https://doi.org/10.1038/s41596-022-00685-8

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