- Authors: Biswas J. et al.
- Year: 2021
- Journal: STAR Protoc 2
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
- Name: SV40 Mouse embryonic fibroblasts
- Name: U-2 OS
Description: Human bone osteosarcoma
Known as: U2OS
- Name: HEK-293T
Mouse embryonic fibroblasts (MEFs) and human osteosarcoma (U2OS) cells are cultured in DMEM 4.5 g glucose, glutamine, supplemented with 1% penicillin streptomycin (P/S) and 10% heat-inactivated fetal bovine serum. For adherent MEFs or U2Os cells we use triplicate 10 cm dishes (70–80% confluence gives best results). Follow best practices for transfection reagent of choice. Optional: Before beginning next step, check transfection efficiency of each dish using GFP fluorescence. Additionally, for mCherry ADAR control plasmids, check RFP fluorescence Optional: If transfection efficiency is high (> 75%) it is possible to omit the following FACS sorting step and proceed directly to RNA isolation (Eg. HEK cell transfection).
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).