Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.


Statistics
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability For data statistical analysis we used SPPSS software; for image reconstructions we used Partseq program (freely available at: https://4dnucleome.cent.uw.edu.pl/PartSeg/), Amira (ThermoFisher), Imaris (Bitplane) and Chimera (UCSF). For the defining subdomains within the 3D structures found, we utilized our custom made Python scripts, which are provided together with the manuscript, available from Github repository, https://github.com/3DEMISH/3D-EMISH.
3D-EMISH image processing code and data files are available at the following public 456 repository server: https://github.com/3DEMISH/3D-EMISH nature research | reporting summary

October 2018
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. On the basis of the aforementioned studies, we estimated a sufficient number of cellular nuclei that allowed for quantitative analysis of nuclear sructure changes, by which we expected that a similar statistical sample is needed for chomatin structure characterization. The particular statistical sample size was restriced from the top by the capacity of the imaging system.
Some of the images were excluded after visual inspection that showed defects related to the imperfect cutting of the sample by the diamond knife There were two biological replicates in the study. All attempts were succsessful.
There was no treatment group, therefore a randomization was not relevant to our study.
Blinding was not relevant to our study, because of its descriptive rather than comparative nature.