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| Status |
Public on Apr 26, 2022 |
| Title |
Y5p9 AAS bulk |
| Sample type |
SRA |
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| Source name |
s288c
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
treatment: AAS
strain: s288c
sample type: bulk
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| Treatment protocol |
After two washes in the S medium by centrifugation (500 g, 5 min) at room temperature, spheroplasts were re-suspended to 5 ODU/ml in the desired treatment solution: amino acids starvation (AAS): S medium (with 1.0 M Sorbitol) without amino acid; Cells were exposed to the treatment for at least 30 min (up to 5 hours) before harvest.
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| Growth protocol |
A monoclone of the yeast strain S288C (ATCC) was selected using a YPD based agar (10% yeast extract, 20% peptone, 2% glucose and 20% agar) petri plate and stocked at -80 °C until use. To wake up cells, 30 µl thawed yeast stock inoculated in 3 ml YPD medium (1% yeast extract, 2% peptone and 2% glucose) was incubated overnight at 30 °C and 250 rpm. Cells were then expanded at 30 °C and 250 rpm after a 1:50 dilution in the YPD medium until mid-logarithmic phase (OD600 between 0.5 and 0.8). Five OD unit (ODU) cells were collected by centrifugation (500 g, 5 min) at room temperature. The cells were resuspended in autoclaved water and collected by centrifugation (500 g, 5 min) at room temperature. The cells were then resuspended in the softening medium (100 mM Hepes-KOH, pH 9.4, 10 mM Dithiothreitol) and incubated in room temperature for 15 min. The cells collected by centrifugation (500 g, 5 min) at room temperature were then resuspended in the Spheroplasts (S) medium (1× YNB, 2% glucose, 1x amino acids, 50 mM Hepes-KOH, pH 7.2, and 1 M sorbitol) [66] to a concentration of 5 ODU/ml. Zymolyase 100T was added to the spheroplasts suspension to a final concentration of 2 μl/ODU, followed by 60 min incubation at 30°C to remove the cell wall.
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| Extracted molecule |
total RNA |
| Extraction protocol |
0.5 mL of the spheroplasts were placed on a poly-lysine coated circular cover slip (2 mm diameter) in a petri dish for 5 min at room temperature (23 °C). The cover slip was broken in the center by a forceps, and a small piece of cover slip was transferred to a 30 µl perfusion chamber, which was constantly perfused by a desired solution by gravity feeding. The solution change time in the chamber was about 20 sec. Single cells were harvested using a path clamp electrode pipette using a micromanipulator (ROE-200, Sutter) under an inverted microscope (Olympus 1X71) placed on a vibration isolation table (TMC). A cell was harvested in less than 10 nl perfusion solution.
Bulk mRNA was extracted from population spheroplasts under AAS using a yeast RiboPureTM RNA Purification Kit (Amion). Different amount of purified bulk mRNA (5pg, 10pg, 20pg, 1,00pg, 1000pg and 10,000pg) were used to construct sequencing libraries in the same way as for single-cell libraries, with the exception that 0.1μl ERCC spike-in mRNA (Thermo Fisher) was added to the lysis buffer, with a concentration of 1:5x105, 1:2.5x105, 1:1.25x105, 1:2.5x104, 1:2.5x103 1:2.5x102 for the 5pg, 10pg, 20pg, 100pg 1,000pg and 10,000pg input RNA, respectively.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The raw reads from FASTQ files of different lanes were first combined based on sample ID
Reads were then mapped to the S. cerevisiae reference genome using STAR (version 2.5.2) without trimming.
Gene transcription levels were quantified using uniquely mapped reads in TPM(transcript per million mapped reads) by RSEM(v1.2.31) and in raw counts by HTSeq
Assembly: SGD R64-2-1
Supplementary files format and content: Tab-separated values files
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| Submission date |
Apr 23, 2022 |
| Last update date |
Apr 27, 2022 |
| Contact name |
Yangqi Su |
| E-mail(s) |
[email protected]
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| Phone |
7043450214
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| Organization name |
UNC Charlotte
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| Department |
Bioinformatics
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| Street address |
4142 E Morada Ln, Apt 6303
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| City |
Stockton |
| State/province |
CA |
| ZIP/Postal code |
95212 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (2) |
| GSE201385 |
Gene expression profile of yeast cells under stress [bulk RNA-seq] |
| GSE201387 |
Gene expression profile of yeast cells under stress |
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| Relations |
| BioSample |
SAMN27750591 |
| SRA |
SRX14981005 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6062433_Y5p9_QC.geneCounts.formatted.for.DESeq.txt.gz |
21.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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