RNA-seq of Saccharomyces cerevisiae - PE2: 30C and 4 hours of industrial fermentation - Experiment 2
Samples of S.  cerevisiae PE-2 were harvested directly from Eldorado mill from Odebrecht  Agroindustrial S/A (Atvos) at Rio Brilhante-MS, located in the center-west  region of Brazil. These experiments were conducted after 6 months of harvest.  The fermentation was carried out using a batch-fed system, with molasse from  sugarcane with 18 Brix of sugar, due to the cell recycling after the end of  the fermentation batch, the yeasts were recovered by centrifugation for reuse  in the subsequent fermentation cycle. To evaluate the effect of temperature  on the yeast gene expression, the temperature of one of the fermentation  tanks was controlled, to operate close to 32 C and the other vat was  maintained at temperatures above 38 C. The samples were collected 4 and 8  hours after the beginning of feeding of the tanks, representing the medium  and the end of the fermentation, respectively. After the process, all samples  were immediately frozen. The total RNA of the samples was extracted using a  phenol and chloroform protocol [Hoffman, 2001]. Illumina RNA-Seq libraries  were prepared following the manufacturers protocol. Briefly, mRNA was  isolated from 1 g of total RNA using oligo(dt) magnetic beads and fragmented  in the presence of divalent zinc ions. The fragmented RNA was then used for  first and second strand cDNA synthesis. Double-stranded cDNA was end-repaired  and 3 adenylated for the ligation of sequencing adapters. After adapter  ligation, fragments of approximately 250 bp were isolated by gel electrophoresis  and PCR amplified. The libraries were validated on an Experion DNA chip  (Bio-Rad, Hercules, CA, USA) and quantified using a Qubit fluorometer  (Invitrogen, Carlsbad, CA, USA). Each library was sequenced in one lane of an  Illumina Genome Analyzer IIx (GAIIx) sequencer, which produced 20-30 million  36-bp single-end reads.