Illumina HiSeq 2500 sequencing; GSM1386606: Fast cycling condition time point 1; Saccharomyces cerevisiae; RNA-Seq

Fast cycling condition time point 1

The synthetic cost of cycling genes is higher than other genes, and the cyclic expression pattern of these genes is a strategy for reducing the overall energy usage of cells Overall design: Samples for both conditions were taken over two metabolic cycles. For the fast cycling condition one sample was taken every 13 minutes for ~4.25 hours. For the slow cycling condition, samples were taken every 36 minutes for ~14.5 hours. Cycling genes were identified using JTK_Cycle (Hughes et al. (2010) Journal of Biological Rhythms).

Cycling transcriptional networks reduce the synthetic cost of genomes

Submitted by Gene Expression Omnibus on 19-NOV-2015

Approximately 2.0 x 108 cells per time interval were rapidly harvested from the fermentor and frozen at -80°. The 44 samples were randomly assigned to four different groups for RNA harvest. The cells from each sample were lysed by adding 200 μLof 425-600 μm glass beads (Sigma-Aldrich) and 700 μLof RNA Stat-60 (amsbio) to the cell pellet and disrupting the mixture with a Mini-Beadbeater-24 (BioSpec Products) in five, 30 second intervals with 2 minutes on ice between each interval. 200 μLof chloroform was added to the disrupted cells, and the samples were centrifuged for 15 minutes (12,000 x g) at 4°C. Subsequently, 500 μLof the aqueous layer was collected, and 200 μL isopropanol was added to this. After incubating at room temperature for 10 minutes, the samples were centrifuged for 10 minutes (12,000 x g) at 4°C, and the supernatants were discarded. The pellets were washed once with 100% and again with 75% molecμLar biology grade ethanol. The pellets were air dried and resuspended in 200 μL water. The quality of the total RNA was verified on a Bioanalyzer RNA 6000 Nano chip (Agilent). The total RNA was spiked in with 1 μL of ERCC RNA Spike-In Mix at 1:30 dilution (Ambion). Subsequently, mRNA pull-down was performed using  NEXTflex™ Poly(A) Beads as per manufacturer’s instructions (Bioo Scientific). The removal of ribosomal RNAs was confirmed on a Bioanalyzer RNA 6000 Pico chip using the mRNA assay (Agilent). Stranded mRNA libraries were prepared using in-house enzyme mixes and buffers. 20 ng of mRNA was fragmented by chemical fragmentation at 95°C for 10 minutes. Fragmented RNA was reverse transcribed using Enzscript™ (M-MLV Reverse Transcriptase Rnase H-) (Enzymatics), followed by directional second strand synthesis. Subsequently, adenylation was performed and then adapter ligation. The adapters were ordered from Bioo Scientific. Uracil DNA Glycosylase treatment was performed prior to amplification by PCR.  The quality of the libraries was checked on a Bioanalyzer High Sensitivity DNA chip (Agilent) and the libraries were quantified by qPCR using Kapa library quantification kit (Kapa Biosystems)

Cycling transcriptional networks reduce the synthetic cost of genomes

Submitter Supplied Information

Samples