RNAi.

RNAi interference (RNAi) was used to generate loss-of-function RNAi phenotypes by feeding nematodes E. coli strain HT115 (DE3) expressing double-stranded RNA (dsRNA) homologous to a target gene (67,–69). RNAi was carried out as described previously (70). Briefly, E. coli with appropriate vectors was grown in LB broth containing ampicillin (100 μg/ml) and tetracycline (12.5 μg/ml) at 37°C overnight and plated onto nematode growth medium (NGM) plates containing 100 mg/ml ampicillin and 6 mM isopropyl-β-d-thiogalactoside (IPTG) (RNAi plates). RNAi-expressing bacteria were allowed to grow overnight at 37°C. Gravid adults were transferred to RNAi-expressing bacterial lawns and allowed to lay eggs for 8 h. Two generations of RNAi were performed. RNAi targeting unc-22 was included as a positive control to account for the RNAi efficiency. Because RNAi for let-653 is larval lethal, RNAi was performed on stage 4 larvae (L4). All RNAi clones except mul-1 were from the Ahringer RNAi library (Open BioSource). The mul-1 clone was obtained from the Vidal RNAi library (Open BioSource). All clones were sequenced using universal GeneWiz M13 primers.