Single-cell m6A profiling in the mouse brain uncovers cell type-specific methylomes and age-dependent differential methylation [m6A]

N6-methyladenosine (m6A) is an abundant mRNA modification in the brain which plays important roles in neurodevelopment and brain function. However, due to technical limitations, global profiling of m6A sites within the individual cell types that make up the brain has not been possible. Here, we develop a mouse model that enables transcriptome-wide m6A detection in any tissue of interest and at single-cell resolution. We use these mice to map m6A across different brain regions and within single cells of the mouse cortex and discover a high degree of shared methylation across brain regions and cell types. However, we also identify a small number of differentially methylated mRNAs in neurons that encode important regulators of neuronal signaling, and we discover that microglia have lower levels of m6A compared to other cell types. Finally, we perform single-cell m6A mapping in aged mice and identify many transcripts with age-dependent changes in m6A. Mice expressing tamoxifen-inducible APOBEC1-YTH were used to identify sites of m6A methylation across three different brain regions in bulk. Since the APOBEC1-YTH transgene induces C-to-U mutations at sites of m6A methylation, these raw files consist of RNA-seq data. Sequening data from APOBEC1-YTHmut expressing mice are used in the m6A site calling process as a background control for C-to-U edits. Additionally, whole cortexes from APOBEC1-YTH and APOBEC1-YTHmut were used for single-cell RNA-seq. Split-pool barcoding (Parse Biosciences) was used to generate these libraries and they were sequenced as a pool, with cellular barcodes used to identify replicates and transgene expression.