Characterization of suppressed lines

Four P₀ L4 larvae were placed in each of 35 mm OP50 NGM plates and allowed to lay embryos for 24 hr at the experimental temperature (15°, 20°, or room temperature). Worms were then transferred to new plates and returned to experimental temperature to continue laying embryos for 24 hr. The number of embryos and hatched animals on overnight plates was counted on each plate, and plates were incubated for 24 hr. The following day, the number of unhatched embryos or hatched larvae was counted, and percent hatching was quantified.

Unless otherwise stated, five L1 stage worms per strain were fed at 20° (Timmons et al. 1998). Animals were moved to new RNAi feeding plates after reaching the L4 stage, and hatching was quantified daily for 48 hr. Worms at the L1 stage were moved onto NGM plates with ampicillin and isopropyl-β-D-thiogalactopyranoside, which were seeded with HT115(DE3) bacteria carrying RNAi feeding constructs, for 24 hr. Worms were then moved onto new RNAi feeding plates daily and hatching embryos were counted.

Two-hundred young adult worms were grown at 20° on 100 mm OP50-seeded plates for one generation and collected by washing in M9 buffer. Each worm pellet was resuspended in 1× SDS loading buffer (2 μl/mg of pellet) and heated in a microwave (4 × 20 sec with 1 min cooling). Lysates were then centrifuged (15,000 × RCF, 10 min) and supernatant was transferred into new tubes. Next, 10 µl of worm lysate was loaded per well and analyzed by standard western blot. SEP-1 was detected by using a polyclonal rabbit antibody (Richie et al. 2011) at a dilution of 1:750. Actin was detected using the mouse monoclonal antibody C4 from Millipore (Temecula, CA) at a dilution of 1:5000. Secondary antibodies used were anti-rabbit 700 and anti-mouse 700 from Li-Cor. Quantification was done using Image Studio software. Actin was used to normalize signals between lanes. All antibody incubations were done in the presence of 5% (w/v) nonfat milk.

Twenty-five males generated using him-5 RNAi bacterial feeding for each strain were mated with five hermaphrodites on unseeded NGM plates and incubated at 20° for 24 hr. Mated worms were then moved to OP50-seeded 60 mm plates and allowed to lay F₁ embryos at 15°. Once F₁ worms reach L4 stage and the presence of ∼50% male animals was observed, indicative of successful mating, four L4 hermaphrodites in triplicate were moved to OP50-seeded 35 mm NGM plates and incubated at 20°. Viability of F₂ embryos was determined.