|
| Status |
Public on Mar 25, 2024 |
| Title |
sef1_YPGly_repeat 2 |
| Sample type |
RNA |
| |
|
| Source name |
log-phase sef1 deletion mutant cells, YPGly, 30°C, 5.5 h, biological repreat 2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303ScSef1NA1
|
| Treatment protocol |
Cells from overnight cutures were subsequently diluted into YPD and YPGly in a cell density of 0.2 OD600/ml, incubated for 5.5 h at 30°C, and harvested.
|
| Growth protocol |
Cells were grown in the YPD medium at 30°C overnight.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted by phenol-chloroform followed by ethanol precipitation. gDNA contaminations wete removed by the TURBO DNA-free kit (AM1907, Ambion). The final RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was checked with the Agilent 2100 Bioanalyzer.
|
| Label |
Alexa Flour® 555
|
| Label protocol |
For Alexa dye-labeled cDNA synthesis, the kit SuperScriptTM Plus Indirect cDNA Labeling System (L101402, Invitrogen by Thermo Fisher Scientific) was used according to the manufacturer's instruction. Briefly, 10 mg of DNase-treated RNA were reverse-transcribed using the anchored oligo[(dT)20-VN] primers and the cDNA was labeled with aminoallyl-dUTP followed by a column purification (28106, QIAquick DNA Purification Kit, Qiagen). The aa-cDNA was then labeled with Alexa Flour® 555 dye (A-32756, Invitrogen by Thermo Fisher Scientific) and purified with the QIAGEN column (28106, QIAquick DNA Purification Kit, QIAGEN).
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| |
|
| Hybridization protocol |
The Agilent Gene Expression Hybridization Kit (5188-5242, Agilent Technologies) was used for hybridization according to the manufacturer's instruction. Briefly, 16 ml of Alexa dye labeled cDNA in water was denatured at 98°C for 3 min in a reaction volume of 40 ml containing 4 ml Agilent 10x blocking Agent and 20 ml of Agilent 2x GEx Hybridization Buffer HI-RPM. Denatured dye-labeled cDNAs were subsequently hybridized to a Agilent Yeast V2 gene expression 8x15K microarray (Design ID: 016322, Agilent Technologies) at 65°C and rotated at 10 rpm on an Agilent hybridizer for 17 h. After hybridization, the microarrays were washed using the Agilent Gene Expression Wash Kit (5188-5327, Agilent Technologies). Briefly, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 and 1 min with 37°C GE Wash buffer 2, and then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned at 535 nm (for Alexa Flour® 555 dye) on the Agilent DNA Microarray Scanner (US9230696) using one color scan setting for 8x15k array slides (scan area: 61 x 21.6 mm; scan resolution: 5 mm; dye channel: green; green PMT: 100%; tiff: 16 bit; XDR: 0.10).
|
| Description |
Gene expression after 5.5 h of subculture
|
| Data processing |
Scanned images were analyzed with the Feature Extraction 10.5.1.1 Software (Agilent Technologies) using default parameters (protocol: GE1_105_Dec08 and grid: 023206_D_F_20131222; provided by the IMB Genomics Core Lab, Academia Sinica, Taiwan) to obtain the background subtracted and spatially detrended Processed Signal intensities. Features flagged in the Feature Extraction Software as the feature non-uniform outlierswere excluded.
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| |
|
| Submission date |
Mar 20, 2024 |
| Last update date |
Mar 25, 2024 |
| Contact name |
Po-Chen Hsu |
| E-mail(s) |
godshi2006@gmail.com
|
| Organization name |
Academia Sinica
|
| Department |
Institute of Molecular Biology
|
| Lab |
N411 JYL lab
|
| Street address |
128 Academia Road, Section 2, Nankang
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE262043 |
Sef1-dependent gene expression in Saccharomyces cerevisiae |
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