RNAi induction was performed as described (Kamath et al., 2001). Cultures of bacteria containing RNAi vectors from the RNAi feeding library generated by J. Ahringer at the University of Cambridge (Geneservice, Cambridge, UK) were grown in LB supplemented with 50 μg/mL ampicillin for 12 - 18 hours at 37°C with shaking. Nematode Growth Medium (NGM) plates containing 1mM IPTG and 25 μg/mL ampicillin were seeded with bacteria containing RNAi vectors, and were incubated at room temperature for 24 hours. 3-5 L4 stage wild-type N2 or sodh-1(ok2799) worms were placed on the seeded plates and incubated at 20°C for 36-40 hours. Adult worms were moved to new RNAi plates and allowed to lay eggs for 1-2 hours. Plates were incubated at 20°C to allow worms to develop to adulthood. First day adults were collected and subjected to allyl-alcohol survival assays. Knockdown of target gene expression levels was confirmed using quantitative RT-PCR (Table 2). Primers used in quantitative RT-PCR experiments are in Supplemental Table 1.
