title: NextSeq 500 paired end sequencing; RNA-seq of yeast cell cycle, used for absolute gene expression quantification
design: RNA-seq of yeast cell cycle, used for absolute gene expression quantification
library_construction_protocol: 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 in 2 L culture flasks and incubated at 30°C, 200 rpm for the duration of sample collection. For transcriptomic samples, 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.  The yeast Saccharomyces cerevisiae CEN.PK113-7D (MATa, MAL2–8c, SUC2 ∆bar1::KANMX) was used. Three individual colonies were inoculated into 10 mL Delft minimal medium respectively and grown 2 x overnight (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. 0.05. Once cells had reached an OD600 of approx. 0.2, alpha-factor was added to a final concentration of 15 ng/mL and incubated for 3 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).
attributes.tag: Experimental Factor: time
attributes.value: 109
attributes.tag: Experimental Factor: time point
attributes.value: 7