|
| Status |
Public on Jun 04, 2020 |
| Title |
0.25 nM, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
haploid yeast mating type MATa
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: yAA95 (ura3::pAA35(PFUS1-Ubi(I)-sfGFP-TFUS1 :URA3) bar1delta::kanMX6 mfalpha1delta::SpHIS3 mfalpha2delta::hphNT1 arg4delta::klTRP1)
treatment: alpha-pheromone, 0.25 nM
time: 60 min
replicate: 2
|
| Treatment protocol |
At mid-exponential phase (OD600 approximately 0.5), pheromone stimulation was initiated by transferring aliquots of the cell suspension into separate flasks with prepared stock solutions of synthetic alpha-pheromone (Sigma). After incubation for 60 minutes cells were harvested by transferring 5 ml of suspension into 15-ml tubes containing 5 ml ice and mixed to immediately cool down the suspension. Further steps were carried out on ice or at 4°C. After centrifugation and removal of supernatant, cell pellets were re-suspended in ice-cold water and transferred to 2-ml Eppendorf tubes. After another centrifugation step the supernatant was carefully removed and the cell pellets were stored at -20°C until further processing.
|
| Growth protocol |
Strain yAA95 was grown in a glass-flask at 30°C and 200 rpm shaking with SD medium composed of yeast nitrogen base (YNB, Formedium) with complete supplement mix (CSM, Formedium) and 2% glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets were re-suspended in 500 µl ProtK buffer (100 mM Tris/Cl pH 7.9, 150 mM NaCl, 25 mM EDTA, 1% SDS) with freshly added 100 µg/ml Proteinase K (ThermoFisher). 500 µl glass beads were added and cells were disrupted by strong vortexing for 5 minutes. The resulting lysate-glass bead mixture was incubated for 60 minutes at 37°C. The RNA was isolated by 25:24:1 aqua-phenol:chloroform:isoamyl alcohol (Carl Roth) extraction followed by a chloroform extraction and precipitated with ethanol. The RNA pellet was washed once with 75% ethanol, re-suspended in water and treated with RNase-free DNaseI (Roche Life Science) according to the manufacturer’s protocol. Prior to library construction, the RNA was depleted of ribosomal RNAs with the Ribo-Zero Gold rRNA Removal Kit (Illumina) and reverse-transcribed with random hexamers
Multiplexed library preparation of total RNA and deep sequencing with IlluminaHiSeq 2000 technology with a read length of 50 bases was performed at EMBL gene core facility (Heidelberg, Germany).
Directional RNA-Seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RPKM_YEAST_pheromone_2.txt
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to SacCer3 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of gene per Megabase of library size (RPKM) were calculated. The number of reads falling in the gene of this meta-transcript were counted and normalized by the size of the transcript and by the size of the library.
Genome_build: SacCer3
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...
|
| |
|
| Submission date |
Jun 03, 2020 |
| Last update date |
Jun 04, 2020 |
| Contact name |
Victor Sourjik |
| E-mail(s) |
victor.sourjik@mpi-marburg.mpg.de
|
| Organization name |
Max Planc Institute for Terrestrial Microbiology
|
| Department |
Systems and Synthetic Microbiology
|
| Street address |
Karl-von-Frisch-Straße 10
|
| City |
Marburg |
| State/province |
Hessen |
| ZIP/Postal code |
35043 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13821 |
| Series (1) |
| GSE151729 |
RNA-sequencing study of global gene expression in haploid yeast Saccharomyces cerevisiae mating type MATa treated with different doses of alpha-pheromone |
|
| Relations |
| BioSample |
SAMN15095597 |
| SRA |
SRX8465091 |
