Abstract
We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.
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Data availability
All raw sequencing data used in this study are available through the European Nucleotide Archive (https://www.ebi.ac.uk/ena/data/view/PRJEB23327). The processed data can be downloaded from the Mendeley Data repository (https://doi.org/10.17632/wbk8hk87r2.3). The test data used in the Walkthrough section of this protocol can be downloaded from github (https://github.com/deLaatLab/mc4c_test-data/archive/master.zip) and are included as Supplementary Data 2.
Code availability
The MC-4C pipeline (version 1.02, at the moment of protocol preparation), including all scripts used to produce figures in this protocol, is available under MIT License at github in mc4c_py repository (https://github.com/deLaatLab/mc4c_py) and is available directly as Supplementary Data 1. The data and results presented in this protocol were prepared using version 1.02 of the MC-4C pipeline.
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Acknowledgements
We thank E. Vos and E. deWit and other laboratory members for their input at various stages of the development of MC-4C. This work was supported by an NWO VIDI grant (639.072.715) to J.d.R and an NWO/CW TOP grant (714.012.002) and NWO VICI grant (724.012.003) to W.d.L., and by the NIH Common Fund Program, grant U01CA200147, as a Transformative Collaborative Project Award (TCPA; TCPA-2017-DE-LAAT).
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Contributions
C.V. and B.A.M.B. designed and performed experiments. C.V. and A.A. wrote manuscript and designed the figures. A.A. designed and performed the computational analysis, designed the figures and wrote the manuscript. P.H.L.K., M.J.A.M.V. and C.V.-Q. performed ‘C’ methods experiments. R.S. and A.A. implemented the pipeline in Python. I.J.R. performed and W.P.K. designed and supervised MinION sequencing experiments. G.G. helped with computational analysis. J.d.R. designed and supervised the computational analyses and pipelines and co-wrote the manuscript. W.d.L. conceived and supervised the study and wrote the manuscript.
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Competing interests
C.V., B.A.M.B., P.H.L.K., M.J.A.M.V. and G.G. are shareholders of Cergentis. W.d.L. is founder and shareholder of Cergentis. W.P.K. and J.d.R. are co-founders and shareholders of Cyclomics.
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Peer review information Nature Protocols thanks Jian Ma 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
Allahyar, A. et al. Nat. Genet. 50, 1151–1160 (2018): https://doi.org/10.1038/s41588-018-0161-5
Integrated supplementary information
Supplementary Fig. 1 gRNA template.
5 μl of gRNA template PCR was loaded on a 2% agarose gel stained with ethidium bromide.
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Supplementary Manual
Supplementary Data 1
MC-4C pipeline
Supplementary Data 2
MC-4C test dataset
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Vermeulen, C., Allahyar, A., Bouwman, B.A.M. et al. Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies. Nat Protoc 15, 364–397 (2020). https://doi.org/10.1038/s41596-019-0242-7
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DOI: https://doi.org/10.1038/s41596-019-0242-7
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