Injections were performed as in [1]. Templates for dsRNA synthesis were made by PCR on L4440-based feeding constructs using T7 primer (5'-CGTAATACGACTCACTATAG-3'). Sense and antisense RNAs were synthesized in a single reaction in vitro using a T7 polymerase-based kit (Promega). Double-stranding was achieved by incubation at 72°C for 10 min, and the sizes of dsRNA products were verified by electrophoresis. dsRNA was injected at a concentration of 0.5-1.0 mg/ml into one or both gonad arms (we and others have found that injection into one or both gonad arms produces equivalent effects). Injected worms were allowed to recover at 22°C for 24 h post-injection, then were replica plated and allowed to lay eggs for 24 h. Injected worms and their progeny were scored as previously described for RNAi by feeding.
