Spatially resolved m6A profiling using m6A-ARTR-DBiT.

N6-methyladenosine (m6A) on RNA plays diverse regulatory roles, yet its spatial distribution within tissues remains largely unexplored. Here we introduce m6A-ARTR-DBiT, a spatial m6A profiling assay that leverages reverse-transcription-based detection and deterministic barcoding in tissue to map transcriptome-wide m6A distribution while preserving native tissue context. Applying m6A-ARTR-DBiT to mouse embryonic tissues and adult brains generates spatially resolved m6A landscapes and reveals region-associated m6A features across different functional domains. Pairwise comparison of spatial m6A profiles with spatial transcriptomes uncovers positive correlations between m6A levels and the expression of its methyltransferases and binding proteins, which also enables systematic identification of tissue-region-specific epitranscriptomic regulation. In the mouse hippocampus, m6A-ARTR-DBiT allows for high-resolution mapping of m6A organization within fine-scale tissue structures. Together, m6A-ARTR-DBiT provides a platform for interrogating RNA modification distribution within intact tissue sections, offering insights into the link between spatially patterned m6A deposition and gene regulation.