Global Yeast Extraction Protocol

1) The yeast species Saccharomyces cerevisiae were cultured in SD media with 2% raffinose. Before extraction, yeast were precultured, diluted to 0.2 OD₆₀₀ units/ml and cultured to 2 OD units/ml, washed and stored at -80^oC.

2) Yeast cells were resuspended in 150 mM NH₄HCO₃  (pH 8) and disrupted by zirconia beads (0.5 mm; BioSpec Products).

3) Yeast cell lysates were diluted to 0.2 OD units (≈2*10⁶ cells) per 200 μL, and mixed with 20μL of internal lipid standard mixture.

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4) Samples were extracted with 990 μL chloroform/methonal (17:1 V:V) for 120 min. The lower organic 17:1 phase lipid extract was collected.

5) The remaing aqueous sample material was reextracted with 990μL chloroform/methonal (2:1 V:V) for 120 min. The lower organic 2:1 phase lipid extract was collected. Then the lipid extracts were vacuum evaporated.

6) The lipid extracts were dissolved in 100 μL chloroform/methonal (1:2 V:V).