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

Cell Rep Methods. 2021 Sep 27;1(5):100061. doi: 10.1016/j.crmeth.2021.100061. Epub 2021 Aug 2.

Abstract

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 m6A-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell m6A modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular m6A levels as CD34+ leukemic progenitors differentiate to CD14+ myeloid cells. We then coupled single-molecule m6A detection with fluorescence in situ hybridization (FISH) to relate mRNA and m6A 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34
  • In Situ Hybridization, Fluorescence*
  • RNA, Messenger / genetics

Substances

  • RNA, Messenger
  • Antigens, CD34