RNA-seq and Ribo-seq upon RNAi in C. elegans

We investigated how RNA interference contends with ongoing translation by examining the fate of actively translated mRNAs subjected to RNAi in C. elegans. We performed RNAi in wild-type animals, skih-2 mutants, and skih-2 pelo-1 double mutants to investigate whether SKI (an RNA helicase) and PELOTA (a ribosome rescue factor) have a role in co-translational RNAi. Each library contains a single dsRNA trigger targeting a single gene. In total five triggers were tested in three genes (unc-15, unc-22, and unc-54). We quantified RNA levels and ongoing translation by RNA-seq and Ribo-seq (ribosome footprinting), respectively. For Ribo-seq, we performed two similar but distinct preparations: size selection for "normal" length ribosome footprints (28-30nt), as well as size selection for "short" footprints (15-18nt).