An in vivo parallelized reporter assay to uncover tissue-specific splicing regulatory sequences in a multicellular animal
The PRA unc-16 minigene reporter plasmid library was injected into C.elegans gonads and targeted to neuronal and muscle tissues using rgef1 and myo-3 tissue specific promoters. Each reporter consisted of a random 10 nucleotide (cis-element) sequence in the downstream intron of the cassette exon. The splicing regulatory effect of this element was tested. Each cis-element was paired with a 10 nucleotide barcode downstream of exon 3 which was retained in the final transcript, thus allowing us to trace back reporter RNA to the reporter plasmid it originated from. These plasmids were sequenced to assess library composition and identify unique cis-element barcode pairs (Reads in Plasmid fastq files). The RNA was collected following injections and sequenced (Reads in cDNA fast files). A subset of elements were re-injected, acting as a validation of the obtained results (Results in small in vivo PRA files 230105_miseq data).