Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.
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Data have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE162357.
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We thank P. Faber of the University of Chicago Genomics Facility for sequencing support and Q. Jin for helping with UHPLC–QQQ–MS/MS. We also thank T. Wu, H.L. Shi and Z.J. Zhang for discussions. L.H. was supported by a Chicago Fellows Program, Chicago Biomedical Consortium (CBC) postdoctoral award and a Leukemia & Lymphoma Society Special Fellow Award. B.T.H. was supported by an NIH fellowship (F32 CA221007). We thank support from National Institutes of Health (NIH) grants RM1 HG008935 (C.H.), R01 GM126553 (M.C.), R01 CA243386 (J.C.), R01 CA214965 (J.C.), R01 CA236399 (J.C.), R01 CA211614 (J.C.) and R01 DK124116 (J.C.), The Simms/Mann Family Foundation (J.C.) and The Margaret Early Medical Research Trust (R.S.). M.C. is supported by a Sloan Foundation Research Fellowship and a Human Cell Atlas Seed Network grant from the Chan Zuckerberg Initiative. C.H. is an investigator of the Howard Hughes Medical Institute. J.C. is a Leukemia & Lymphoma Society (LLS) Scholar.
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. The remaining authors declare no competing interests.
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Hu, L., Liu, S., Peng, Y. et al. m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome.
Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01243-z