In Vitro dsRNA Synthesis and RNAi Feeding in C. elegans

The dsRNA was transcribed in vitro from PCR products amplified from pJC53.2 using standard molecular methods (Collins et al. 2010 (link); Rouhana et al. 2013 (link)). Concentration of dsRNA was determined using either a NanoPhotometer NP80 (Implen, Munich, Germany) or by band intensity after gel electrophoresis. For a typical experiment, 10–12 animals were fed 1–3-µg dsRNA mixed in ∼30-µL food (beef liver paste, 4:1 liver:salts mixture), and 1-µL green food dye was added to verify that the animals ate. The mixture was doubled for larger experiments. Negative control worms were fed dsRNA matching green fluorescent protein (GFP) or bacterial genes [chloramphenicol resistance gene (Cm^R) and toxin CcdB (ccdB)]. Animals were kept in 60–100-mm Petri dishes. After eating, the animals were washed and transferred to fresh dishes, and salts were supplemented with 1:1000 gentamicin sulfate [50-mg/mL stock (Gemini Bio, West Sacramento, CA)]. Animals were fed dsRNA ∼once per week for 3 total feedings [more feedings given in long-term RNAi experiments (Fig. 2; Supplementary Fig. 6)] and then were processed. Live images during experiments were obtained using a Zeiss Axiocam 506 color camera mounted on a Zeiss Axio Zoom.V16 microscope (ZEISS Microscopy, Jena, Germany). Live images and video were also captured on an iPhone 6 and/or SE and processed in iMovie (Apple Inc., Cupertino, California).