Comprehensive RNAi Screening Protocol

Oligo design, PCR, RNA transcription and annealing, and soaking RNAi were performed as described in Ref. [5] (link). The primer pairs used to amplify 500–1000 bp target regions of candidate genes are listed in Spreadsheet S1. For RNAi experiments, five to seven L4 stage larval worms were soaked in dsRNA (or soaking buffer for control experiments) for 24 hr at 20°C and recovered individually to NGM plates containing 150 µg/ml Nile Blue A, which penetrates permeable, but not intact, eggshells [4] (link). dsRNA-treated adults were allowed to lay embryos for 24 hr at 25°C (Plate 1), then transferred to a fresh plate to lay embryos for an additional 24 hr (Plate 2). One day after the adult was removed, the number of hatched larvae, impermeable embryos (clear), and permeable embryos (blue) were counted for each plate, the sum of which was recorded as brood size. Averages for the 5–7 worms are reported (Spreadsheet S1.xlsx). A maximum of 50 hatched larvae per plate were counted. Control worm plates normally contained 0–4% permeable embryos.