Yeast strains and growth media

Yeast strains and growth media

Yeast strains

-    Revive strains of interest from -80°C glycerol stock onto YPD agar plates (10 g/L yeast extract, 20 g/L bacteriological peptone, 20 g/L bacteriological agar, 55 g/L glucose monohydrate)

-    Grow for 2 overnights at 30°C, and store plates at 4°C until use

Growth media

All experiments are performed at 30°C using the following rich media:

-     YPMAL10%:
Autoclave YP2x solution (20 g/L yeast extract, 40 g/L bacteriological peptone) and filter-sterilize MAL20% (210.5 g/L maltose monohydrate). Mix ½ of YP2x with ½ of MAL20% to reach desired concentrations

-    YPGLU5%:
Autoclave YP2x solution (20 g/L yeast extract, 40 g/L bacteriological peptone) and filter-sterilize GLU20% (220 g/L glucose monohydrate). Mix ½ of YP2x with ¼ of autoclaved dH₂O and ¼ of GLU20% to reach desired concentrations

Growth conditions specifically for scRNA-seq experiment

-    Pre-grow cells in 3 mL YPMAL10% in dilute conditions for 1 overnight at 30°C in a spinning wheel, making sure final cell counts remain under 2*10⁶ cells/mL

-    Refresh media and make serial dilution so that after the 2^nd overnight (30°C, spinning wheel), cultures are at a cell count of ~2*10⁶ cells/mL

-    Wash and inoculate cell cultures into 50 mL YPGLU5% to reach a starting cell density of 10⁴ cells/mL and grow for 12 hr (shaking incubator, 30°C – 200 rpm)

-   Wash to YPMAL10% and monitor cell counts continuously throughout lag phase

-  For each sample taken during the lag phase, immediately freeze 0.5 mL of culture with 0.5 mL of ice-cold glycerol50% at -80°C until the running day of the Chromium device