Fission yeast strains and general techniques

Cells were grown in 180 ml of EMM liquid medium in a 500-ml flask at 30°C with continuous shaking at 170 rpm until the cell density reached OD600 = 0.2. A 45-ml aliquot of the culture was taken for the “0 min” sample and fixed by mixing with 5 ml solution of trichloroacetic acid (TCA; final concentration = 10%). The remaining culture was then filtered onto a mixed cellulose ester membrane (φ 47 mm, pore size 0.45 µm; Advantec, USA), and the harvested cells on the membrane were rinsed with 100 ml of EMM-N (EMM without NH₄Cl) to initiate nitrogen starvation. The washed cells were immediately resuspended in pre-warmed 135 ml of EMM-N liquid medium in a 500-ml flask and incubated at 30°C with continuous shaking at 170 rpm. At each time point after nitrogen starvation, a 45-ml aliquot of the culture was harvested and fixed with 5-ml TCA.

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