C. elegans cultures from approximately 150 10 cm diameter NGM
plates were collected and egg-prepped using hypochlorite bleach solution
to isolate eggs from gravid hermaphrodites. Eggs were allowed to
hatch overnight in M9 and a synchronized population of L1 larva was
transferred to liquid culture medium and allowed to grow at 20 °C
until the population reached the young adult stage. Worms were collected
and washed three times to generate an approximately 8 mL
dense pellet. For lipid droplet proteomics analysis, the pellet and all solutions
were spiked with protease inhibitor cocktail solution (Sigma
Aldrich P2714). Worms were manually chopped with razor blade tool
(10–12 new razor blades taped together and sterilized with ethanol)
on an ice cold glass plate until visually all the worms had been broken
open. The worm pellet was then dounce homogenized 40 plunges
with a tight fitting pestle. Pellet was spun at 200 ×g at 4 °C for 5 min
and then an equal volume of 1.08 M sucrose solution was added and
spun at 2000 g for 5 min to remove large debris. Supernatant was
transferred to a 13.2 mL thinwall ultraclear centrifuge tube (Beckman-
Coulter 344059) and layered with a gradient of 0.27 M sucrose,
0.135 M sucrose and Top solution buffer (25 mM Tris–HCl; 1 mM
EDTA; 1 mM EGTA; protease inhibitor cocktail). Spun in SW41 rotor
using a Beckman L8-70M ultracentrifuge at 35,000 rpm for 30 min at
4 °C. The top layer (white, cloudy) was transferred via a glass pipet to
a siliconized microcentrifuge tube and spun at 18,000 ×g for 10 min
and the bottom, aqueous layer was removed