Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution

Epigenetic modifications control the stability and translation of mRNA molecules. Here, we present a microscopy-based platform for quantifying modified RNA molecules and for relating the modification patterns to single-cell phenotypes. We directly capture mRNAs from cell lysates on oligo-dT-coated coverslips, then visually detect and sequence individual m(6)A-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell m(6)A modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular m(6)A levels as CD34(+) leukemic progenitors differentiate to CD14(+) myeloid cells. We then coupled single-molecule m(6)A detection with fluorescence in situ hybridization (FISH) to relate mRNA and m(6)A levels of individual genes to single-cell phenotypes. This single-cell multi-modal assay suite can empower investigations of RNA modifications in rare populations and single cells.