Synchronized animals were raised at 20 °C, unless otherwise noted, and fed OP50 E. coli bacteria until they reached adulthood. For each strain, animals were collected with M9 solution at day 1 of adulthood for analysis. For RNAi experiments, wild-type (N2, WT) and glp-1(e2141) animals were raised at 25 °C on OP50 E. coli bacteria and were transferred onto plates freshly seeded with control bacteria or bacteria expressing dsRNA against the gene of interest. Animals were incubated at 20 °C for 48 h, then collected and washed twice with M9 solution.
Nematodes (~1,000) were flash frozen in liquid N2 and RNA was extracted with Trizol (Invitrogen/Life Technologies, Carlsbad, CA) as described38 (link). Concentration and purity of RNA samples were determined with a NanoDrop spectrophotometer and samples were stored at −80 °C. Reverse transcription was performed on 1 μg RNA per sample using iScript Supermix (Bio-Rad, Hercules, CA). Samples were diluted 1/100 and complementary DNA standards (1/25–1/400) were prepared as serial dilutions from a mixture of the relevant cDNAs. Diluted samples and custom-designed primers (IDT, San Diego, CA) were mixed with SYBR Green (Roche, Indianapolis, IN) and samples were analysed using a Roche LightCycler 480 (Roche). Relative mRNA levels of target genes were normalized against the geometric mean39 (link) of the housekeeping genes act-1, cyn-1, cdc-42 and pmp-3. Primer sequences can be found in Supplementary Table S10. Nematode orthologues of the TFEB target genes analysed in this study were selected based on their significance in previous studies8 (link),14 (link). Each biological sample was analysed in duplicate or triplicate for each gene assayed. The mRNA levels of nematode genes are presented as mean±s.d. and statistical analysis of biological triplicates was performed by two-tailed Student’s t-test or analysis of variance using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA).