Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.
Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA; Department of Neurobiology, Duke University School of Medicine, Durham, NC 27710, USA.
STAR Protoc. 2022 Aug 19;3(3):101646. doi: 10.1016/j.xpro.2022.101646. eCollection 2022 Sep 16.
Most techniques for mapping mA-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 mA 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 mA sites. For complete details on the use and execution of this protocol, please refer to Tegowski et al. (2022).
大多数绘制 mA 甲基化 RNA 转录组图谱的技术都需要大量的 RNA，并且仅限于批量细胞和组织。在这里，我们提供了一种使用单细胞 DART-seq（scDART-seq）在单个 HEK293T 细胞中鉴定 mA 位点的详细方案。该方案详细说明了如何生成诱导表达 APOBEC1-YTH 转基因的细胞系，以及如何使用重要的对照来最小化假阳性。我们还描述了用于鉴定 mA 位点的生物信息学分析。有关此方案的使用和执行的完整详细信息，请参阅 Tegowski 等人。（2022 年）。
