Quantification of Fatty Acid Profiles

Fatty acid contents were measured as previously described [50 (link),59 (link)]. 20,000 age-synchronized L4 worms were washed off plates and washed three times with water. Worm pellets were resuspended with 1.2 mL 2.5% H₂SO₄ in methanol and incubated at 80°C for 1 hour. Then, 1ml supernatants were mixed with 1.2 ml hexane and 1.8 ml water to extract fatty acid methyl esters for GC-MS/MS analysis. The Supelco 37 Component FAME Mix (Sigma Aldrich) was used to determine the retention time. The Shimadzu GCMS-TQ8040 Gas Chromatograph Mass Spectrometer equipped with a SH-Rxi-5sil MS column was used. Fatty acid contents were normalized to protein concentrations.