Abstract
The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.
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Data availability
All processed Nodewalk data have been deposited in the Gene Expression Omnibus (GSE76049). The ChIP and DamId-seq data were retrieved from the Gene Expression Omnibus as follows: cLADs (GSE22428), H3K9me2 (GSE58534) and H3K4me1 (GSM1240111). Source data for Figs. 6, 7, 8 and 3, 4 are available online in refs. 24,25, respectively. The images in Fig. 2 represent unprocessed original Bioanalyzer files.
Code availability
The analysis pipeline is available at https://github.com/Anita-Rolf-lab/Nodewalk.
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Acknowledgements
This work was supported by the Swedish Research Council (VR 03108), the Swedish Childhood Cancer Fund (PR2017-0132), the Swedish Cancer Society (CAN 708), the Lundberg Foundation (2018-0138), Karolinska Institutet, the Novo Nordisk Foundation (NNF16OC0021512), the Cancer Society in Stockholm (2018–2021) and the KA Wallenberg Foundation (KAW 2017.0077). We thank A. F. Woodbridge for his efforts in the initial development of the Nodewalk pipeline and R. Ohlsson for valuable discussions.
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J.V. updated the Nodewalk protocol and contributed to manuscript writing; N.S. contributed to the invention of the Nodewalk assay, developed the entire protocol and contributed to the development of the analysis pipeline and manuscript writing; R.M. and D.B. designed the submitted state of pipeline and implemented the Python code; D.B. designed the manual and tested the code; S.W. contributed to the pipeline code integration and A.G. contributed to the invention of the Nodewalk assay and wrote the manuscript.
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D.B. has formed a bioinformatics company, Genomiki Solution Ltd., that analyses high throughput data, including chromatin fibre interaction maps.
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Nature Protocols thanks Argyris Papantonis 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
Sumida, N. et al. Nucleic Acids Res. 48, 10867–10876 (2020): https://doi.org/10.1093/nar/gkaa817
Scholz, B. A. et al. Nat. Genet. 51, 1723–1731 (2019): https://doi.org/10.1038/s41588-019-0535-3
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Vestlund, J., Sumida, N., Mehmood, R. et al. The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations. Nat Protoc 18, 755–782 (2023). https://doi.org/10.1038/s41596-022-00774-8
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DOI: https://doi.org/10.1038/s41596-022-00774-8
