In Situ Hi-C Library Preparation for Plants to Study Their Three-Dimensional Chromatin Interactions on a Genome-Wide Scale

Part of the Methods in Molecular Biology book series (MIMB, volume 1629)


The spatial organization of the genome in the nucleus is critical for many cellular processes. It has been broadly accepted that the packing of chromatin inside the nucleus is not random, but structured at several hierarchical levels. The Hi-C method combines Chromatin Conformation Capture and high-throughput sequencing, which allows interrogating genome-wide chromatin interactions. Depending on the sequencing depth, chromatin packing patterns derived from Hi-C experiments can be viewed on a chromosomal scale or at a local genic level. Here, I describe a protocol of plant in situ Hi-C library preparation, which covers procedures starting from tissue fixation to library amplification.

Key words

In situ Hi-C Plants Chromatin packing 



This work was supported by Deutsche Forschungsgemeinschaft (LI 2862/1).


  1. 1.
    Dekker J, Rippe K, Dekker M, Kleckner N (2002) Capturing chromosome conformation. Science 295:1306–1311CrossRefPubMedGoogle Scholar
  2. 2.
    Zhao Z, Tavoosidana G, Sjolinder M, Gondor A, Mariano P, Wang S, Kanduri C, Lezcano M, Sandhu KS, Singh U, Pant V, Tiwari V, Kurukuti S, Ohlsson R (2006) Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38:1341–1347CrossRefPubMedGoogle Scholar
  3. 3.
    Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J (2006) Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Res 16:1299–1309CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Simonis M, Kooren J, de Laat W (2007) An evaluation of 3C-based methods to capture DNA interactions. Nat Methods 4:895–901CrossRefPubMedGoogle Scholar
  5. 5.
    Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326:289–293CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Feng S, Cokus SJ, Schubert V, Zhai J, Pellegrini M, Jacobsen SE (2014) Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis. Mol Cell 55:694–707CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Grob S, Schmid MW, Grossniklaus U (2014) Hi-C analysis in Arabidopsis identifies the KNOT, a structure with similarities to the flamenco locus of Drosophila. Mol Cell 55:678–693CrossRefPubMedGoogle Scholar
  8. 8.
    Wang C, Liu C, Roqueiro D, Grimm D, Schwab R, Becker C, Lanz C, Weigel D (2015) Genome-wide analysis of local chromatin packing in Arabidopsis thaliana. Genome Res 25:246–256CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Burton JN, Adey A, Patwardhan RP, Qiu R, Kitzman JO, Shendure J (2013) Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol 31:1119–1125CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Marie-Nelly H, Marbouty M, Cournac A, Flot JF, Liti G, Parodi DP, Syan S, Guillen N, Margeot A, Zimmer C, Koszul R (2014) High-quality genome (re)assembly using chromosomal contact data. Nat Commun 5:5695CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680CrossRefPubMedGoogle Scholar
  12. 12.
    Nagano T, Varnai C, Schoenfelder S, Javierre BM, Wingett SW, Fraser P (2015) Comparison of Hi-C results using in-solution versus in-nucleus ligation. Genome Biol 16:175CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ferretti L, Sgaramella V (1981) Specific and reversible inhibition of the blunt end joining activity of the T4 DNA ligase. Nucleic Acids Res 9:3695–3705CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sexton T, Yaffe E, Kenigsberg E, Bantignies F, Leblanc B, Hoichman M, Parrinello H, Tanay A, Cavalli G (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148:458–472CrossRefPubMedGoogle Scholar
  15. 15.
    Folta KM, Kaufman LS (2006) Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat Protoc 1:3094–3100CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Center for Plant Molecular Biology (ZMBP)University of TübingenTübingenGermany

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