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Mapping RNA–chromatin interactions by sequencing with iMARGI


RNA–chromatin interactions represent an important aspect of the transcriptional regulation of genes and transposable elements. However, analyses of chromatin-associated RNAs (caRNAs) are often limited to one caRNA at a time. Here, we describe the iMARGI (in situ mapping of RNA–genome interactome) technique, which is used to discover caRNAs and reveal their respective genomic interaction loci. iMARGI starts with in situ crosslinking and genome fragmentation, followed by converting each proximal RNA–DNA pair into an RNA–linker–DNA chimeric sequence. These chimeric sequences are subsequently converted into a sequencing library suitable for paired-end sequencing. A standardized bioinformatic software package, iMARGI-Docker, is provided to decode the paired-end sequencing data into caRNA–DNA interactions. Compared to its predecessor MARGI (mapping RNA–genome interactions), the number of input cells for iMARGI is 3–5 million (a 100-fold reduction), experimental time is reduced, and clear checkpoints have been established. It takes a few hours a day and a total of 8 d to complete the construction of an iMARGI sequencing library and 1 d to carry out data processing with iMARGI-Docker.

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Fig. 1: iMARGI protocol and linker design.
Fig. 2: Computational workflow of iMARGI data analysis.
Fig. 3: Checkpoints for nuclear integrity.
Fig. 4: Size distributions of intermediate and final products from HEK293T cells.
Fig. 5: Checkpoints and troubleshooting.
Fig. 6: Visualization of iMARGI data in GIVE.

Data availability

An iMARGI dataset has been deposited into the NCBI Sequence Read Archive (SRA) under accession no. SRR8206679.

Code availability

The iMARGI-Docker software and its documentation are available at: The software is completely open source, under the BSD 2 license. The source code is available at The pre-built Docker image can be pulled from the Docker Hub. The version used in this paper is v.1.1.1.


  1. 1.

    Nguyen, T. C., Zaleta-Rivera, K., Huang, X., Dai, X. & Zhong, S. RNA, Action through Interactions. Trends Genet. 34, 867–882 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. & Brockdorff, N. Requirement for Xist in X chromosome inactivation. Nature 379, 131–137 (1996).

    CAS  Article  Google Scholar 

  3. 3.

    Yang, F. et al. The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation. Genome Biol. 16, 52 (2015).

    Article  Google Scholar 

  4. 4.

    Graf, M. et al. Telomere length determines TERRA and R-loop regulation through the cell cycle. Cell 170, 72–85.e14 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Yu, R., Wang, X. & Moazed, D. Epigenetic inheritance mediated by coupling of RNAi and histone H3K9 methylation. Nature 558, 615–619 (2018).

    CAS  Article  Google Scholar 

  6. 6.

    Watanabe, T. et al. Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332, 848–852 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Miao, Y. et al. Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function. Nat. Commun. 9, 292 (2018).

    Article  Google Scholar 

  8. 8.

    Place, R. F., Li, L. C., Pookot, D., Noonan, E. J. & Dahiya, R. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc. Natl Acad. Sci. USA 105, 1608–1613 (2008).

    CAS  Article  Google Scholar 

  9. 9.

    Morris, K. V., Chan, S. W., Jacobsen, S. E. & Looney, D. J. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305, 1289–1292 (2004).

    CAS  Article  Google Scholar 

  10. 10.

    Rinn, J. L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007).

    CAS  Article  Google Scholar 

  11. 11.

    Simon, M. D. et al. The genomic binding sites of a noncoding RNA. Proc. Natl Acad. Sci. USA 108, 20497–20502 (2011).

    CAS  Article  Google Scholar 

  12. 12.

    Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. & Chang, H. Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol. Cell 44, 667–678 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Engreitz, J. M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013).

    Article  Google Scholar 

  14. 14.

    Sridhar, B. et al. Systematic mapping of RNA-chromatin interactions in vivo. Curr. Biol. 27, 602–609 (2017).

    CAS  Article  Google Scholar 

  15. 15.

    Yan, Z. et al. Genome-wide co-localization of RNA-DNA interactions and fusion RNA pairs. Proc. Natl Acad. Sci. USA 116, 3328–3337 (2019).

    CAS  Article  Google Scholar 

  16. 16.

    Bell, J. C. et al. Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts. eLife 7, e27024 (2018).

    Article  Google Scholar 

  17. 17.

    Li, X. et al. GRID-seq reveals the global RNA–chromatin interactome. Nat. Biotechnol. 35, 940–950 (2017).

    CAS  Article  Google Scholar 

  18. 18.

    Chen, W. et al. RNAs as proximity-labeling media for identifying nuclear speckle positions relative to the genome. iScience 4, 204–215 (2018).

    CAS  Article  Google Scholar 

  19. 19.

    Yin, Y. et al. U1 snRNP regulates chromatin retention of noncoding RNAs. Preprint at bioRxiv, (2018).

  20. 20.

    Sun, G. et al. A bias-reducing strategy in profiling small RNAs using Solexa. RNA 17, 2256–2262 (2011).

    CAS  Article  Google Scholar 

  21. 21.

    Jayaprakash, A. D., Jabado, O., Brown, B. D. & Sachidanandam, R. Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 39, e141 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Sorefan, K. et al. Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 3, 4 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Li, W., Freudenberg, J. & Miramontes, P. Diminishing return for increased Mappability with longer sequencing reads: implications of the k-mer distributions in the human genome. BMC Bioinforma. 15, 2 (2014).

    Article  Google Scholar 

  24. 24.

    Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Hadjiolov, A. A., Tencheva, Z. S. & Bojadjieva-Mikhailova, A. G. Isolation and some characteristics of cell nuclei from brain cortex of adult cat. J. Cell Biol. 26, 383–393 (1965).

    CAS  Article  Google Scholar 

  26. 26.

    Suzuki, K., Bose, P., Leong-Quong, R. Y., Fujita, D. J. & Riabowol, K. REAP: a two minute cell fractionation method. BMC Res. Notes 3, 294 (2010).

    Article  Google Scholar 

  27. 27.

    Nabbi, A. & Riabowol, K. Isolation of nuclei. Cold Spring Harb. Protoc. 2015, 731–734 (2015).

    PubMed  Google Scholar 

  28. 28.

    Andrews, S. FastQC: a quality control tool for high throughput sequence data. (2010).

  29. 29.

    Dekker, J. et al. The 4D nucleome project. Nature 549, 219–226 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Quinlan, A. R. & Hall, I. M. J. B. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

  31. 31.

    Abdennur, N. & Mirny, L. Cooler: scalable storage for Hi-C data and other genomically-labeled arrays. Bioinformatics (2019).

  32. 32.

    Kerpedjiev, P. et al. HiGlass: web-based visual exploration and analysis of genome interaction maps. Genome Biol. 19, 125 (2018).

    Article  Google Scholar 

  33. 33.

    Cao, X., Yan, Z., Wu, Q., Zheng, A. & Zhong, S. GIVE: portable genome browsers for personal websites. Genome Biol. 19, 92 (2018).

    Article  Google Scholar 

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We thank X. Wen for help with graphics; 4DN DCIC ( for discussions on the data processing pipeline; A. Zheng, A. Kaul, K. Faizi, N. Moshiri, R. Zhang, J. Ma, J. Chen, X. Huang and Z. Zhang for testing the iMARGI-Docker software; and K. Sriram and A. Chen for proofreading. This work was funded by DP1HD087990 (to S.Z.), NIH 4D Nucleome U01CA200147 (to S.C. and S.Z.), and NIH R00HL122368 and R01HL145170 (to Z.C.).

Author information




W.W. and S.Z. designed the research; W.W., T.C.N. and S.Z. developed the experimental method and protocol; Z.Y. and S.Z. developed the computational method and data analysis tools; W.W., Z.Y. and S.Z. wrote the manuscript; Z.C. and S.C. revised the manuscript.

Corresponding author

Correspondence to Sheng Zhong.

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Competing interests

S.Z. is a cofounder of Genemo Inc.

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Peer review information Nature Protocols thanks Michiel de Hoon and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Sridhar, B. et al. Curr. Biol. 27, 602–609 (2017)

Yan, Z. et al. Proc. Natl Acad. Sci. USA 116, 3328–3337 (2019)

Chen, W. et al. iScience 4, 204–215 (2018)

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Wu, W., Yan, Z., Nguyen, T.C. et al. Mapping RNA–chromatin interactions by sequencing with iMARGI. Nat Protoc 14, 3243–3272 (2019).

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