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| Status |
Public on Apr 26, 2022 |
| Title |
Y700 isotonic single-cell |
| Sample type |
SRA |
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| Source name |
s288c
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
treatment: isotonic
strain: s288c
sample type: single-cell
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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; glucose starvation (GS): S medium (with 1.0 M Sorbitol) without glucose; hypotonic: S medium without sorbitol; isotonic condition: S medium with 1M sorbitol. 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.
A harvested cell was quickly transferred using a home-made microinjection system to a 200 µl Eppendorf tube containing 4 µl cell lysis buffer (0.9× PCR Buffer II, 3 mM MgCl2, 0.45% NP40, 4.5 mM DTT, 0.18 U/μl SUPERase-In, 0.36 U/μl RNase Inhibitor, 12.5 nM AUP1 primer, 2 mM dNTP). In most single-cell samples, 0.1 μl (1:2.5x104 dilution) ERCC spike-in mRNA (Thermo Fisher) was added. The cell was lysed at 70 °C for 90 sec, then placed on ice and stored at -80 °C until use. A cell lysate was thawed on ice, and 1 μl reverse transcription mix was added (13.2 U/ μl SuperScript III Reverse transcriptase, 0.4 U/μl Rnase Inhibitor, and 0.07 μg/μl T4 gene 32 protein). The first strand cDNA was synthesized by incubating the tube at 50 °C for 30 min, followed by inactivation of the reverse transcriptase at 70 °C for 10 min, and then the tube was cooled on ice. Free AUP1 primers were removed by adding 1 µl ExoSAP (Affymetrix) to the tube and incubating at 37 °C for 15 min, followed by inactivation of the ExoSAP at 80 °C for 15 min. This step would leave the AUP1 sequences at the 5’-end cDNA intact. A polyA tail was then added to the 3’-end of the first strand cDNA by adding 6 µl TdT mixture (1× PCR Buffer II, 1.5 mM MgCl2, 3 mM dATP, 0.75 U/μl Terminal Transferase and 0.1 U/μl RNase H) and incubating at 37 °C for 15 min, followed by inactivation of the enzyme at 70 °C for 10 min. The resulting products (12 μl) were then divided into two equal portions (each 6 μl), and each was mixed with 19 μl second strand buffer (1× High Fidelity PCR Buffer, 2 mM MgSO4, 0.2 mM each dNTP, 0.3 μM AUP2 primer, and 0.1 U/μl high fidelity Platinum Taq DNA polymerase). The two tubes were subject to one PCR cycle (30 sec at 95 °C, 2 min at 50 °C and 6 min at 72 °C) to synthesize the second-strand cDNA in the form of 5’-AUP2-T24-cDNA-A24-AUP1-3’. Nineteen μl PCR mixture (1× High Fidelity PCR Buffer, 2 mM MgSO4, 0.25 mM each dNTP, 2 μM AUP1 Primer, 2 μM AUP2 Primer, 0.1 U/μl Platium Taq DNA Polymerase High Fidelity) was added to each tube, which brings the volume of each reaction to 44 μl, and cDNA was amplified by 18 PCR cycles (98 °C for 5 sec, 67 °C for 1 min and 72 °C for 6 min). The resulting cDNA from two reactions were combined (total 88 μl) and were further subject to 12 cycles of PCR with two duplicates, each with 2.4 μl sample and 87.6 μl PCR mixture (1× High Fidelity PCR Buffer, 2 mM MgSO4, 0.375 mM each dNTP, 1 μM AUP1 Primer, 1 μM AUP2 Primer, 0.1 U/μl Platium Taq DNA Polymerase High Fidelity). The products were then combined, and cDNA was revolved on a 1% ager gel (25 μl sample per lane). The band between 300 bases to the loading well was cut and cDNA was purified using a QIA quick gel purification Kit, followed by magnetic beads (GE Health) purification (10:7 sample to beads ratio). After quantification using a Bioanalyzer (Agilent High Sensitivity DNA Kit), the libraries were then prepared using an Illumina Nextera XT or TruSeq DNA Sample Preparation Kit according to the vender’s guide. The libraries were sequenced on an Illumina HiSeq2000 or HiSeq2500 machine (100 base-paired reads). Bulk mRNA was also 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 single cell |
| 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
Differential expression was performed by MAST
promotor motifs were found using Prosampler
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) |
| GSE201386 |
Gene expression profile at single cell level yeast cells under stress |
| GSE201387 |
Gene expression profile of yeast cells under stress |
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| Relations |
| BioSample |
SAMN27750669 |
| SRA |
SRX14981145 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6062532_Y700_QC.geneCounts.formatted.for.DESeq.txt.gz |
17.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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