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m6A-SAC-seq for quantitative whole transcriptome m6A profiling

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

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

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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.

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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.

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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).

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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).

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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

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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.

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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.

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Key reference using this protocol

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

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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

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