Department of Anatomy and Structural Biology, Bronx, NY 10461, USA.
Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, MA 02451, USA.
STAR Protoc. 2021 Jun 30;2(3):100634. doi: 10.1016/j.xpro.2021.100634. eCollection 2021 Sep 17.
Targets of RNA-binding proteins discovered by editing (TRIBE) determines RNA-proteins interactions and nuclear organization with minimal false positives. We detail necessary steps for performing mammalian cell RBP-TRIBE to determine the targets of RNA-binding proteins and MS2-TRIBE to determine RNA-RNA interactions within the nucleus. Necessary steps for performing a TRIBE experiment are detailed, starting with plasmid/cell line generation, cellular transfection, and RNA sequencing library preparation and concluding with bioinformatics analysis of RNA editing sites and identification of target RNAs. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020).
RNA 结合蛋白编辑（TRIBE）发现的靶标可确定 RNA-蛋白质相互作用和核组织，假阳性结果最少。我们详细介绍了进行哺乳动物细胞 RBP-TRIBE 以确定 RNA 结合蛋白的靶标和 MS2-TRIBE 以确定核内 RNA-RNA 相互作用的必要步骤。详细介绍了进行 TRIBE 实验的必要步骤，从质粒/细胞系的生成、细胞转染、RNA 测序文库的制备开始，最后进行 RNA 编辑位点的生物信息学分析和靶 RNA 的鉴定。有关本协议使用和执行的完整详细信息，请参阅 Biswas 等人（2020 年）。
