Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution.
Cell Rep Methods
; 1(5)2021 09 27.
Article
in En
| MEDLINE
| ID: mdl-34734208
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
In Situ Hybridization, Fluorescence
Type of study:
Diagnostic_studies
Language:
En
Journal:
Cell Rep Methods
Year:
2021
Document type:
Article
Affiliation country:
Estados Unidos