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SRX5486144: RNA-Seq of Naumovia castelii in log growth phase in rich media
1 ILLUMINA (Illumina HiSeq 2500) run: 24.4M spots, 2.4G bases, 1.1Gb downloads

Design: Starting with a frozen cell pellet from our experiment, RNA extraction was performed using the MasterPure RNA Extraction Kit protocol; QC measures were taken using Nanodrop, EtBr gel, and Bioanalyzer. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 (ref. RS-122-2101/2) according to the manufacturer's protocol. 1g of total RNA were used for poly(A)-mRNA selection using streptavidin-coated magnetic beads. Synthetic spike-in transcripts (ERCC RNA Spike-In Mix 1) were added to each sample and the mixture was subsequently fragmented to approximately 300bp (range 180 500bp). cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Double-stranded DNA was purified with AMPure XP beads and was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. Purification of the final library was done with AMPure beads. Library amplification was performed by 15 cycles of PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Caliper LabChip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illuminas cBot. Libraries were then sequenced on the Illumina HiSeq 2500, Paired End, 50nts using v4 chemistry, in 1 lane, pool of 12 libraries.
Submitted by: Hospital del Mar Research Institute
Study: Transcriptomic comparison of 11 species of yeast in rich media and oxidative stress conditions
show Abstracthide Abstract
The objective of this experiment was to study the mechanism of de novo gene birth in Saccharomyces cerevisiae. While the model organism S. cerevisiae has one of the highest quality reference genomes and annotations, other members of Saccharomycotina are not so fortunate. We designed our experiment to compare the transcriptomes of 11 species of yeast in 'normal' as well as 'stress' conditions as it has been hypothesized that de novo genes could play a role in the evolution of environmental stress responses. We used this RNA sequencing data to assemble reference-independent transcriptomes for each species to avoid biases in our comparative transcriptomic analysis caused by varying qualities of reference annotations.We grew 11 species (S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, N. castelii, K. lactis, L. waltii, L. thermotolerans, L. kluyveri, and Schizo. pombe) in two parallel conditions:1. Rich medium- We isolated isogenic populations from streaked plates, grew cultures overnight in culture tubes, then inoculated 20mL of a custom rich medium* in 50mL Erlenmeyer flasks at 30ºC. Cells were harvested in log growth phase at an OD600 of approximately 0.25, then subsequently frozen at -80ºC.2. Oxidative stress- The same isogenic populations were grown in parallel, identical to the first 'Rich medium' condition; however, 30 minutes prior to harvesting the cells, diluted hydrogen peroxide was added to a final concentration of 1.5mM.* The custom rich medium, taken from the following study, was designed to accommodate growth of many species of yeast: Tsankov AM, Thompson DA, Socha A, Regev A, Rando OJ. “The role of nucleosome positioning in the evolution of gene regulation”. PLoS Biol 2010.
Sample: N. castelii oxidative stress
SAMN11070714 • SRS4458821 • All experiments • All runs
Name: n_castellii_stress-ATTCCT
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Selection: PolyA
Layout: PAIRED
Runs: 1 run, 24.4M spots, 2.4G bases, 1.1Gb
Run# of Spots# of BasesSizePublished


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