Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Nature Protocols volume 8, pages 176–189 (2013)Cite this article

Subjects

Abstract

N6-methyladenosine–sequencing (m6A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m6A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation–sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m6A between and within gene transcripts. When applied to human and mouse transcriptomes, m6A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m6A. The protocol can be completed within ∼9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic diagram of the m6A-seq protocol (Steps 1–28).
Figure 2: Bioinformatic pipeline.
Figure 3: Calibration of RNA fragmentation and validation of size distribution.
Figure 4: Deduction of methylation consensus motifs and evaluation of their location relative to summits of m6A peaks.
Figure 5: Representative human gene plots harboring m6A peaks.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Cantara, W.A. et al. The RNA Modification Database, RNAMDB: 2011 update. Nucleic Acids Res. 39, D195–D201 (2011).

    Article  CAS  Google Scholar 

  2. He, C. Grand challenge commentary: RNA epigenetics? Nat. Chem. Biol. 6, 863–865 (2010).

    Article  CAS  Google Scholar 

  3. Chan, C.T. et al. A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet. 6, e1001247 (2010).

    Article  CAS  Google Scholar 

  4. Schaefer, M. et al. RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. Genes Dev. 24, 1590–1595 (2010).

    Article  CAS  Google Scholar 

  5. Bokar, J. Fine-tuning of RNA functions by modification and editing. in Topics in Current Genetics 12 (ed. Grosjean, H.) 141–177 (Springer, 2005).

    Article  CAS  Google Scholar 

  6. Zhong, S. et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 20, 1278–1288 (2008).

    Article  CAS  Google Scholar 

  7. Clancy, M.J., Shambaugh, M.E., Timpte, C.S. & Bokar, J.A. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 30, 4509–4518 (2002).

    Article  CAS  Google Scholar 

  8. Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    Article  CAS  Google Scholar 

  9. Levanon, E.Y. et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat. Biotechnol. 22, 1001–1005 (2004).

    Article  CAS  Google Scholar 

  10. Klose, R.J. & Bird, A.P. Genomic DNA methylation: the mark and its mediators. Trends Biochem. Sci. 31, 89–97 (2006).

    Article  CAS  Google Scholar 

  11. Dai, Q. et al. Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine. Nucleic Acids Res. 35, 6322–6329 (2007).

    Article  CAS  Google Scholar 

  12. Kellner, S., Burhenne, J. & Helm, M. Detection of RNA modifications. RNA biology 7, 237–247 (2010).

    Article  CAS  Google Scholar 

  13. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  CAS  Google Scholar 

  14. Meyer, K.D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    Article  CAS  Google Scholar 

  15. Czerwoniec, A. et al. MODOMICS: a database of RNA modification pathways. 2008 update. Nucleic Acids Res. 37, D118–121 (2009).

    Article  CAS  Google Scholar 

  16. Horowitz, S., Horowitz, A., Nilsen, T.W., Munns, T.W. & Rottman, F.M. Mapping of N6-methyladenosine residues in bovine prolactin mRNA. Proc. Natl. Acad. Sci. USA 81, 5667–5671 (1984).

    Article  CAS  Google Scholar 

  17. Bringmann, P. & Luhrmann, R. Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6. FEBS Lett. 213, 309–315 (1987).

    Article  CAS  Google Scholar 

  18. Dante, R. & Niveleau, A. Inhibition of in vitro translation by antibodies directed against N6-methyladenosine. FEBS Lett. 130, 153–157 (1981).

    Article  CAS  Google Scholar 

  19. Munns, T.W., Liszewski, M.K., Oberst, R.J. & Sims, H.F. Antibody nucleic acid complexes. Immunospecific retention of N6-methyladenosine-containing transfer ribonucleic acid. Biochemistry 17, 2573–2578 (1978).

    Article  CAS  Google Scholar 

  20. Munns, T.W., Liszewski, M.K. & Sims, H.F. Characterization of antibodies specific for N6-methyladenosine and for 7-methylguanosine. Biochemistry 16, 2163–2168 (1977).

    Article  CAS  Google Scholar 

  21. Munns, T.W., Oberst, R.J., Sims, H.F. & Liszewski, M.K. Antibody-nucleic acid complexes. Immunospecific recognition of 7-methylguanine- and N6-methyladenine-containing 5′-terminal oligonucleotides of mRNA. J. Biol. Chem. 254, 4327–4330 (1979).

    CAS  PubMed  Google Scholar 

  22. Munns, T.W., Sims, H.F. & Liszewski, M.K. Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine. J. Biol. Chem. 252, 3102–3104 (1977).

    CAS  PubMed  Google Scholar 

  23. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  24. Feng, J., Liu, T. & Zhang, Y. Using MACS to identify peaks from ChIP-seq data. Curr. Protoc. Bioinformatics 34, 2.14.1–2.14.14 (2011).

    Article  Google Scholar 

  25. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  Google Scholar 

  26. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Bailey, T.L. & Machanick, P. Inferring direct DNA binding from ChIP-seq. Nucleic Acids Res. 18, 18 (2012).

    Google Scholar 

  29. Salmon-Divon, M., Dvinge, H., Tammoja, K. & Bertone, P. PeakAnalyzer: genome-wide annotation of chromatin binding and modification loci. BMC Bioinformatics 11, 415 (2010).

    Article  Google Scholar 

  30. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  31. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  Google Scholar 

  32. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Kahn Family Foundation for their support. This work was supported in part by grants from the Flight Attendant Medical Research Institute (FAMRI), Bio-Med Morasha Israel Science Foundation (ISF) (grant no. 1942/08), ISF (grant no. 1667/12), the molecular basis of human disease I-CORE (Israeli Centers of Research Excellence) and the Israel Ministry of Science and Technology (Scientific Infrastructure Program). G.R. holds the Djerassi Chair in Oncology at the Sackler Faculty of Medicine, Tel Aviv University. This work was performed in partial fulfillment of the requirements for a PhD degree to D.D., Sackler Faculty of Medicine, Tel Aviv University.

Author information

Author notes

  1. Dan Dominissini and Sharon Moshitch-Moshkovitz: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel

    Dan Dominissini, Sharon Moshitch-Moshkovitz, Mali Salmon-Divon, Ninette Amariglio & Gideon Rechavi

  2. Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

    Dan Dominissini & Gideon Rechavi

Authors
  1. Dan Dominissini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sharon Moshitch-Moshkovitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mali Salmon-Divon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ninette Amariglio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gideon Rechavi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.D. and S.M.-M. conceived the approach, developed the protocol and performed the experiments. M.S.-D. designed the bioinformatic pipeline and analyzed the data. N.A. and G.R. supervised the project. D.D., S.M.-M., M.S.-D., N.A. and G.R. wrote the manuscript.

Corresponding author

Correspondence to Gideon Rechavi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M. et al. Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc 8, 176–189 (2013). https://doi.org/10.1038/nprot.2012.148

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.148

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing