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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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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Author notes

    • Jessica Zuin
    •  & Harmen J G van de Werken

    Present addresses: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland (J.Z.); Department of Urology, Cancer Computational Biology Center, Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, the Netherlands (H.J.G.v.d.W.).


  1. Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.

    • Petros Kolovos
    • , Michael Lesnussa
    • , Jessica Zuin
    • , A M Ali Imam
    • , Harmen J G van de Werken
    • , Kerstin S Wendt
    • , Tobias A Knoch
    •  & Frank Grosveld
  2. Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, the Netherlands.

    • Petros Kolovos
  3. Center for Biomics, Erasmus Medical Centre, Rotterdam, the Netherlands.

    • Rutger W W Brouwer
    • , Christel E M Kockx
    •  & Wilfred F J van IJcken
  4. Chromatin Networks, BioQuant & German Cancer Research Center, Heidelberg, Germany.

    • Nick Kepper


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

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    Supplementary Figure 1 and Supplementary Tables 1–4.

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    Supplementary Data

    Zip file containing the entire T2C analysis pipeline.

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