L4-stage hermaphrodite worms were placed onto NGM plates containing seeded bacteria expressing dsRNA for each gene and were incubated for 36-40 h at 22°C or for 72 h at 15°C. Then, three worms were independently replica plated onto plates seeded with the same bacteria and were allowed to lay eggs for 24 h at 22°C before being removed. Progeny were scored for embryonic lethality after a further 24 h at 22°C, and post-embryonic phenotypes were scored blindly by two independent observers at the end of four successive 12-h intervals. Progeny laid on the first plate were also scored for post-embryonic phenotypes. A gene was found to be positive for a given phenotype if it could be observed in at least two of three worms or their progeny in at least two independent feeding experiments. Feeding times shorter than 36-40 h at 22°C or 72 h at 15°C were not always sufficient to produce a strong RNAi effect; for example, par-2 and par-3 were 53% and 23% embryonic lethal, respectively, after feeding for 24 h but were both 100% embryonic lethal after feeding for at least 36 h at 22°C (our unpublished results).
