m⁶A RNA modifications are measured at single-base resolution across the mammalian transcriptome

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.