Single cell discovery of m6A RNA modifications in the hippocampus

N6-methyladenosine (m6A) is a prevalent and highly regulated RNA modification essential for RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m6A is implicated in neurogenesis and learning. Although extensively studied, its presence and impact in specific cell types remain poorly understood. We investigated m6A in the hippocampus at the single-cell level, revealing a comprehensive landscape of m6A modifications within individual cells. We identified m6A RNA sites in bulk RNA sequencing and in single cell data by detecting RNA editing sites that were introduced near m6A sites. We first tested our approach in HEK293T cells, with bulk RNA-seq. We transfected our samples with YTH viruses that caused editing near m6A sites, while YTHmut viruses and APOBEC1 and wild type samples were used as background controls. After we verified this approach in HEK293T cells, we applied it the mouse hippocampus also with bulk RNA-seq. We then performed single cell sequencing to identify m6A sites in single cells by identifying mutations in the sequencing data. We confirmed our data with m6A-RNA immunoprecipitation and sequencing (m6A-RIP seq).