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

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. Samples are from flow-sorted cortical Neurons. Nuclei were isolated and fixed with ethanol prior to staining with anti-NeuN antibody and flow sorting. Then, total RNA was extracted, and RNA-seq libraries prepared. m6A sites were identified using to Bullseye pipeline to find sites of C-to-U conversion.