scDART-seq reveals distinct m⁶A signatures and mRNA methylation heterogeneity in single cells

N⁶-methyladenosine (m⁶A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m⁶A profiling has revealed important aspects of m⁶A distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m⁶A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m⁶A sites across individual cells and identify differentially methylated mRNAs across the cell cycle. Additionally, we show that cellular subpopulations can be distinguished based on their RNA methylation signatures, independent from gene expression. These studies reveal fundamental features of m⁶A that have been missed by m⁶A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m⁶A deposition.