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:

m6A-SAC-seq for quantitative whole transcriptome m6A profiling

Nature Protocols volume 18, pages 626–657 (2023)Cite this article

Subjects

An Author Correction to this article was published on 22 June 2023

This article has been updated

Abstract

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m6A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233–129,263 sites using as little as 2 ng of poly A+ RNA. These identified sites correspond well with previous m6A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m6A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Overview of the m6A-SAC-seq protocol.
Fig. 2: m6A-SAC-seq can reproducibly identify a large number of m6A sites.
Fig. 3: Quality control of m6A-SAC-seq datasets.

Similar content being viewed by others

Data availability

The underlying data of Figs. 2 and 3 are deposited at GSE198246.

Code availability

Code for assessing the quality, reliability and features of the identified m6A sites is available on GitHub (https://github.com/y9c/m6A-SACseq).

Change history

References

  1. Fu, Y. & He, C. Nucleic acid modifications with epigenetic significance. Curr. Opin. Chem. Biol. 16, 516–524 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Alarcón, C. R., Lee, H., Goodarzi, H., Halberg, N. & Tavazoie, S. F. N6-methyladenosine marks primary microRNAs for processing. Nature 519, 482–485 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Liu, N. et al. N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Res. 45, 6051–6063 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang, X. et al. N6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    Article  PubMed  Google Scholar 

  6. Zhou, K. I. et al. N6-methyladenosine modification in a long noncoding RNA hairpin predisposes its conformation to protein binding. J. Mol. Biol. 428, 822–833 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Liu, J. et al. A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Ping, X.-L. et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, 177–189 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Śledź, P. & Jinek, M. Structural insights into the molecular mechanism of the m6A writer complex. eLife 5, e18434. (2016).

  10. Wang, X. et al. Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex. Nature 534, 575–578 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Wang, Y. et al. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191–198 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wei, C.-M., Gershowitz, A. & Moss, B. Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell 4, 379–386 (1975).

    Article  CAS  PubMed  Google Scholar 

  13. Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. He, P. C. & He, C. m6A RNA methylation: from mechanisms to therapeutic potential. EMBO J. 40, (2021).

  15. Yang, Y., Hsu, P. J., Chen, Y.-S. & Yang, Y.-G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 28, 616–624 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. 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  PubMed  PubMed Central  Google Scholar 

  18. Linder, B. et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Meyer, K. D. DART-seq: an antibody-free method for global m6A detection. Nat. Methods 16, 1275–1280 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, Z. et al. Single-base mapping of m6A by an antibody-independent method. Sci. Adv. 5, eaax0250 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Garcia-Campos, M. A. et al. Deciphering the “m6A code” via antibody-independent quantitative profiling. Cell 178, 731–747.e16 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Shu, X. et al. A metabolic labeling method detects m6A transcriptome-wide at single base resolution. Nat. Chem. Biol. 16, 887–895 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Hu, L. et al. m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nat. Biotechnol. 40, 1210–1219 (2022).

    Article  CAS  PubMed  Google Scholar 

  24. O’Farrell, H. C., Musayev, F. N., Scarsdale, J. N. & Rife, J. P. Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design. Biochemistry 49, 2697–2704 (2010).

    Article  PubMed  Google Scholar 

  25. O’Farrell, H. C., Pulicherla, N., Desai, P. M. & Rife, J. P. Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution. RNA 12, 725–733 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Shu, X. et al. N6-allyladenosine: a new small molecule for RNA labeling identified by mutation assay. J. Am. Chem. Soc. 139, 17213–17216 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Golinelli, M.-P. & Hughes, S. H. Nontemplated nucleotide addition by HIV-1 reverse transcriptase. Biochemistry 41, 5894–5906 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Davis, W. R., Gabbara, S., Hupe, D. & Peliska, J. A. Actinomycin D inhibition of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein. Biochemistry 37, 14213–14221 (1998).

    Article  CAS  PubMed  Google Scholar 

  29. Song, Y., Liu, K. J. & Wang, T.-H. Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture. PLoS ONE 9, e94619 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rusche, J. R. & Howard-Flanders, P. Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase. Nucleic Acids Res. 13, 1997–2008 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y. & Robb, G. B. Structural bias in T4 RNA ligase-mediated 3′-adapter ligation. Nucleic Acids Res. 40, e54–e54 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, K., Peng, J. & Yi, C. The m6A consensus motif provides a paradigm of epitranscriptomic studies. Biochemistry 60, 3410–3412 (2021).

    Article  CAS  PubMed  Google Scholar 

  33. Wei, C.-M. & Moss, B. Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid. Biochemistry 16, 1672–1676 (1977).

    Article  CAS  PubMed  Google Scholar 

  34. Ke, S. et al. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation. Genes Dev. 29, 2037–2053 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang, Y., Xiao, Y., Dong, S., Yu, Q. & Jia, G. Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine. Nat. Chem. Biol. 16, 896–903 (2020).

    Article  CAS  PubMed  Google Scholar 

  36. Zhang, Z. et al. Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library. Nat. Methods 18, 1213–1222 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Zhang, L.-S. et al. Transcriptome-wide mapping of internal N7-methylguanosine methylome in mammalian mRNA. Mol. Cell 74, 1304–1316.e8 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ke, S. et al. m 6 A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 31, 990–1006 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Maden, B. E. H. Locations of methyl groups in 28S rRNA of Xenopus laevis and man: clustering in the conserved core of molecule. J. Mol. Biol. 201, 289–314 (1988).

    Article  CAS  PubMed  Google Scholar 

  40. Maden, B. E. H. Identification of the locations of the methyl groups in 18S ribosomal RNA from Xenopus laevis and man. J. Mol. Biol. 189, 681–699 (1986).

    Article  CAS  PubMed  Google Scholar 

  41. Yang, X. et al. N6-methyladenine modification in noncoding RNAs and its function in cancer. Biomarker Res. 8, (2020).

  42. Mossessova, E. & Lima, C. D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Liu, J. et al. N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367, 580–586 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tegowski, M., Flamand, M. N. & Meyer, K. D. scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells. Mol. Cell 82, 868–878.e10 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank Dr. P. Faber and the University of Chicago Genomics Facility for sequencing support. The authors also thank L. S. Zhang and T. Pan’s lab for discussions. This work was supported by NIH RM1 HG008935, R01CA251150 and the Howard Hughes Medical Institute (C.H. is an investigator of the Howard Hughes Medical Institute).

Author information

Author notes

  1. These authors contributed equally: Ruiqi Ge, Chang Ye.

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Ruiqi Ge, Chang Ye, Yong Peng, Qing Dai, Yutao Zhao, Shun Liu, Pingluan Wang & Chuan He

  2. Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Ruiqi Ge, Chang Ye, Yong Peng, Qing Dai, Yutao Zhao, Shun Liu, Pingluan Wang & Chuan He

  3. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Ruiqi Ge, Chang Ye, Yong Peng, Qing Dai, Yutao Zhao, Shun Liu, Pingluan Wang & Chuan He

  4. Section of Genetic Medicine, Department of Medicine, The University of Chicago, Chicago, IL, USA

    Yong Peng & Shun Liu

  5. Department of Human Genetics, The University of Chicago, Chicago, IL, USA

    Yong Peng & Shun Liu

  6. Fudan University Institutes of Biomedical Sciences, Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, International Co−laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Shanghai Medical College of Fudan University, Shanghai, China

    Lulu Hu

Authors
  1. Ruiqi Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chang Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qing Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yutao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pingluan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lulu Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.G. and C.Y. contributed equally to this work. R.G. developed experimental procedures and performed the experiments. C.Y. developed the analysis pipeline and performed data analysis. Y.P. and S.L. helped in the development of the analysis pipeline and provided valuable discussions on the development of the method. Q.D. synthesized the probes. R.G., Y.Z., Q.D. and P.W. all participated in the synthesis and purification of allyl-SAM. L.H. provided valuable discussions on drafting the manuscript. R.G., C.Y. and C.H. wrote and edited the manuscript.

Corresponding authors

Correspondence to Lulu Hu or Chuan He.

Ethics declarations

Competing interests

A patent application for m6A-SAC-seq has been filed by the University of Chicago. C.H. is a scientific founder and a scientific advisory board member of Accent Therapeutics, Inc. and Inferna Green, Inc.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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

Related links

Key reference using this protocol

Hu, L. et al. Nat. Biotechnol. 40, 1210–1219 (2022): https://doi.org/10.1038/s41587-022-01243-z

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ge, R., Ye, C., Peng, Y. et al. m6A-SAC-seq for quantitative whole transcriptome m6A profiling. Nat Protoc 18, 626–657 (2023). https://doi.org/10.1038/s41596-022-00765-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00765-9

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: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research