T-R3-7
Saccharomyces cerevisiae
Protocols: After incubation with alpha factor, cells were spun for 2 min at 4000 g using a High-Performance Avanti J-26SXP Centrifuge equipped with a JLA-10.500 rotor (Beckman Coulter, USA). Using 100 mL pre-warmed water, cells were washed twice to remove residual alpha factor under the same centrifuge conditions. Cell pellets were then re-suspended in 500 mL pre-warmed Delft minimal medium and incubated at 30°C, 180 rpm for the duration of sample collection. Throughout the entire cell cultivation, care was taken to maximise oxygen availability. For transcriptomic sample collection, cells were added to ice- chilled 50 mL falcon tubes containing approx. 25 mL ice then immediately pelleted in 4°C pre-chilled centrifuge for 3 min at 5500 g. Supernatant was discarded and pellets were snap frozen with liquid nitrogen and stored at -80°C until further notice. The yeast Saccharomyces cerevisiae CEN.PK113-7D (MATa, MAL2-8c, SUC2 [delta]bar1::KANMX) was used. Three individual colonies were inoculated into 5 mL Delft minimal medium respectively, with 2% ethanol used as the carbon source and cells were grown until stationary phase (200 rpm, 30°C). Cultures were then used to inoculate 200 mL Delft medium to an optical density measured at 600 nm (OD600) of approx. 0.04. Once cells had reached an OD600 of approx. 0.3, alpha factor was added to a final concentration of 15 ng/mL and cells were incubated for 5 h (200 rpm, 30°C). RNA was extracted and purified using Qiagen RNeasy Mini Kit extraction and DNA degradation according to the user's manual (Qiagen, Hilden, Germany). Integrity of the product was verified using 2100 Bioanalyzer instrument according to its user's manual (Agilent Technologies, Santa Clara, California, United States). RNA concentration was determined by a NanoDrop 2000 (Thermo Fisher Scientific) and Qubit (Life Technologies). The cDNA libraries were generated by using Illumina TruSeq Stranded mRNA Library Prep (96 Samples) kit (Illumina Inc., USA) with following modifications. Briefly, 2.5 ug of total undegraded RNA was diluted in 50 µL nuclease free water and mRNA was purified by oligo-dT selection by following the company's protocol. Purified mRNA was enzymatically fragmented for 8 minutes to approx. 250 bp average fragment size. Finally, the adapter-modified cDNA fragments were enriched by 10 cycles enrichment PCR. All the purification and size-selection steps along the protocol were conducted by using Kapa Pure Beads (Roche Molecular Systems, Inc., Switzerland).