Detection of m6A in single cultured cells using scDART-seq

STAR Protoc. 2022 Aug 19;3(3):101646. doi: 10.1016/j.xpro.2022.101646. eCollection 2022 Sep 16.

Abstract

Most techniques for mapping m6A-methylated RNAs transcriptome-wide require large amounts of RNA and have been limited to bulk cells and tissues. Here, we provide a detailed protocol for the identification of m6A sites in single HEK293T cells using single-cell DART-seq (scDART-seq). The protocol details how to generate cell lines with inducible expression of the APOBEC1-YTH transgene and the use of important controls for minimizing false positives. We also describe the bioinformatic analysis to identify m6A sites. For complete details on the use and execution of this protocol, please refer to Tegowski et al. (2022).

Keywords: Cell-based Assays; Flow Cytometry/Mass Cytometry; Gene Expression; Molecular Biology; RNAseq; Sequencing; Single Cell.

Publication types

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

MeSH terms

  • APOBEC-1 Deaminase / genetics
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing* / methods
  • Humans
  • RNA
  • Sequence Analysis, RNA / methods
  • Transcriptome* / genetics

Substances

  • RNA
  • APOBEC-1 Deaminase