Targeted RNAi Interference in C. elegans

RNAi interference was performed as described^{6 (link)}. HT115 (DE3) bacteria were transformed with vectors expressing dsRNA targeting the following genes of interest: ash-2, set-2, wdr-5.1, set-16, utx-1, jmjd-3.1, fat-2, fat-5, fat-7, mdt-15, sbp-1, tbh-1, nhr-10, F21A3.11, dod-23 (Ahringer Library, Dr. Andrew Fire), let-363/mTOR (Dr. Xiaomeng Long), daf-15/Raptor (Dr. Allen Hsu), rsks-1, asm-2 and unc-132 (Vidal Library, Dharmacon). All vectors were confirmed by sequencing. Empty vector bacteria, containing the RNAi plasmid without an RNAi insert, served as a control for all RNAi experiments. RNAi culture was concentrated 30-fold, and stored at 4°C for no more than 2 weeks. For double RNAi treatments, two cultures were mixed in a 1:1 ratio by volume. To obtain a synchronized worm population, egglay was performed on appropriate RNAi plates for 1-4 hours. All RNAi treatments began at hatching, except for ORO and lifespan experiments using sbp-1, mdt-15, fat-2, let-363/mTOR, or daf-15/Raptor RNAi, which began at initiation of reproduction (day 2.5 of life) to minimize effects on development.