NextSeq 500 paired end sequencing; RNA-seq of yeast cell cycle grown on glucose, synchronized with CDC15-2.
RNA-seq of yeast cell cycle grown on glucose, synchronized with CDC15-2.
After incubation at 37°C, cells were returned to an air incubator set to 24°C, 180 rpm, and incubated under these conditions for the duration of sample collection. Samples were collected once an hour, over an 8 h time span with the first sample collected immediately after cells were returned to 24°C. 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.PK 113-11A HIS3 TRP1 SPC42tmCherry cdc15-2 was used. Three individual colonies were inoculated into 10 mL Delft minimal medium respectively, with 2% glucose used as the carbon source, and cells were grown 3 x overnight (180 rpm, 24°C). Cultures were then used to inoculate 500 mL Delft medium to an optical density measured at 600 nm (OD600) of approx. 0.025. Once cells had reached an OD600 of approx. 0.08, cultures were switched to another air incubator set to 37°C, 180 rpm, and incubated for 4 h.  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).