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  • Detection of m6A in single ...
    Tegowski, Matthew; Meyer, Kate D.

    STAR protocols, 09/2022, Letnik: 3, Številka: 3
    Journal Article

    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). Display omitted •Generation of transgenic cell lines expressing inducible APOBEC1-YTH•Transgene induction and sorting of individual cells•Preparation of sequencing libraries from single cells•Processing data to identify m6A sites from C-to-U mutations in the sequencing data Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.