Remove lyophilized C. elegans samples from -80°C freezer

Add approximately 200µL of 1.0 mm Zirconia beads (BioSpec Cat. No. 11079110zx) to each sample.

Equipment

Value	Label
FastPrep-96	NAME
High-throughput bead beating grinder and lysis system	TYPE
MP Biomedicals	BRAND
116010500	SKU
http://www.mpbio.com	LINK

Equipment

Value	Label
QuickFlex™ Sample Holder	NAME
Adapter for 96 x 2 mL tube holder on FastPrep-96	TYPE
MP Biomedicals	BRAND
116010570	SKU
http://www.mpbio.com	LINK

Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s

Place samples on dry ice for 0h 1m 30s to avoid overheating due and sample degradation.

Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s

Place samples on dry ice for 0h 1m 30s to avoid overheating due and sample degradation.

Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s

Store on dry ice and proceed with Extraction (Section 2) or store at -80°C until ready to process.

Add 500µL of 100% IPA chilled to -20°C to each homogenized sample containing: (1) lyophilized C. elegans material and (2) zirconia beads

Vortex each sample for 0h 0m 30s to 0h 1m 0s

Let sample sit at Room temperature for 0h 15m 0s to 0h 20m 0s

Add an additional 500µL of 100% IPA chilled to -20°C to each homogenized sample containing: (1) lyophilized C. elegans material, (2) zirconia beads, and (3) 500µL IPA.

Vortex each sample for 0h 0m 30s to 0h 1m 0s

Let sample sit at Room temperature for 0h 15m 0s to 0h 20m 0s

Store samples (~12 hours) at -20°C

Centrifuge samples for 0h 30m 0s at 20800rcf,4°C,0h 0m 0s

Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of non-polar molecules.

Place all non-polar extracts in a CentriVap at Room temperature and monitor until completely dry.

Equipment

Value	Label
CentriVap	NAME
Benchtop Centrifugal Vacuum Concentrator	TYPE
Labconco	BRAND
7810010	SKU
http://www.labconco.com/category/centrivap-cold-traps	LINK

Add 1mL of 80:20 Methanol:Water (MeOH:H₂O) chilled to 4°C to each tube containing (1) the remaining worm pellet and (2) zirconia beads.

Shake samples at 4°C for 0h 30m 0s

Centrifuge samples for 0h 30m 0s at 20800rcf,4°C,0h 0m 0s

Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of polar molecules.

Place all polar extracts in a CentriVap at Room temperature and monitor until completely dry.

Remaining worm pellet and zirconia beads can be stored at -80°C for future protein and/or carbohydrate analysis or discarded appropriately.