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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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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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).
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 (2022). https://doi.org/10.1038/s41596-022-00765-9
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