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Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C

Abstract

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in 7–8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.

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Figure 1: Overview of the T2C procedure.
Figure 2: Flow diagram of the analysis pipeline.
Figure 3: T2C interaction map around SAMD4A at an average 0.4-Kbp resolution.
Figure 4: T2C interaction map for a 3Mb genomic region at an average 0.4-Kbp resolution.

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Acknowledgements

This work was supported by ERASysBio+/FP7 and the following national funding organizations: the Dutch Ministry for Science and Education, the Netherlands Science Organization (NWO), the UK Biotechnology and Biological Sciences Research Council (BSRC) and the German Bundesministerium für Bildung und Forschung (BMBF). P.K. and T.A.K. were supported by grants from EpiGenSys. P.K. was also supported by the NWO (Rubicon fellowship; 019.162LW.011). H.J.G.v.d.W. was supported by a Zenith grant (93511036) from the Netherlands Genomics Initiative. The project was also supported by the Bluescript EU Integrated Project, the SyBOSS EU consortium (no. 050040212), the Netherlands Institute for Regenerative Medicine (NIRM), a MEC Booster grant from the Netherlands Genomics Institute (MEC Booster grant) and a European People Marie Curie Actions Program, Marie Curie European Reintegration Grant (ERG; FP7-PEOPLE-2010-RG).

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Contributions

P.K., C.E.M.K., J.Z., W.F.J.v.I., K.S.W., T.A.K. and F.G. adapted, optimized and troubleshot the protocol for this technique. R.W.W.B., H.J.G.v.d.W. and P.K. performed the bioinformatical analysis, after initial development by M.L., N.K. and T.A.K.; P.K. and F.G. designed the oligonucleotides, with contributions from T.A.K. and A.M.A.I. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Petros Kolovos, Tobias A Knoch or Frank Grosveld.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Example of successful digestion with ApoI.

Agarose gel (1.5%, wt/vol) on which an aliquot of undigested and digested sample is depicted, showing the typical smear of DNA fragments upon digestion with ApoI.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Tables 1–4. (PDF 496 kb)

Supplementary Data

Zip file containing the entire T2C analysis pipeline. (ZIP 192 kb)

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Kolovos, P., Brouwer, R., Kockx, C. et al. Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C. Nat Protoc 13, 459–477 (2018). https://doi.org/10.1038/nprot.2017.132

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