Figure 1. Overview of the HyperTRIBE protocol.

Genetic scheme for building the HyperTRIBE in vivo expression construct. The HyperTRIBE construct is expressed in cultured cells (Steps 1–8) with MT promoter induced by Cu²⁺ (magenta ellipse) or in specific tissues in Drosophila with UAS promoter induced by Gal4 (orange ellipse). In cultured cells, HyperTRIBE construct is cotransfected with an actin-promoter driven GFP plasmid, while UAS driven GFP is co-activated by Gal4 in specific tissues in Drosophila. In this protocol, we only provide procedures for expressing HyperTRIBE construct in cultured cells. The cells expressing the construct together with GFP are sorted by FACS (Steps 9–12) and their transcriptomes are used to prepare RNA-Seq libraries (Steps 28–58). The locations of the nucleotide where the transcripts are edited are marked by I, showing the adenosine to inosine editing by HyperTRIBE construct. Sequencing reads from these libraries are aligned to the transcriptome (Step 78–79), compared to gDNA or wild-type mRNA to identify editing sites and calculate editing percentage (Steps 82–83). We require high confidence editing sites to be present in at least two replicates (Step 84).