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| Status |
Public on Dec 23, 2007 |
| Title |
Spores_incubation in nitrogen_1 hour and 30 minutes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S. cerevisiae spores, 1 hour and 30 minutes after the transfer to nitrogen
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: SK1 wild type
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were flash frozen in liquid Nitrogen. Total RNA was extracted using RNeasy Midi kit (QIAGEN)
|
| Label |
Cy5
|
| Label protocol |
Samples and reference cDNA were synthesized from total RNA and labeled with Cy3 and Cy5 by the indirect amino-allyl method as described below. Total RNA (20 µg) was annealed with 4 µg oligo dT12-18 and reverse transcribed into cDNA with M-MLV Reverse Transcriptase RNase H Minus (Promega) for 2 hours at 45°C in the presence of 2.5mM MgCl2, 0.5 mM of dATP, dCTP and dGTP, 0.1mM dTTP, 0.4mM amino-allyl dUTP. Additional 400U M-MLV Reverse Transcriptase RNase H Minus was added and incubated for another 2 hours. The reaction was terminated by incubation at 70°C for 15 minutes and RNA was degraded by adding 10 µM 1N NaOH and incubation at 65 C for 30 minutes. The sample was neutralized by titrating with 1M HCl. cDNA was purified by ethanol precipitation, resuspended in 10 µl 0.1M Sodium Carbonate buffer pH 9.3. Cy5 or Cy3 were added to experimental or reference sample, respectively, and incubated at 25 C for 1 hour. Labeled cDNA was purified by Strataprep PCR purification kit (Stratagene) according to the manufactures instructions. Dye incorporation was measured using a spectrophotometer.
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| |
|
| Channel 2 |
| Source name |
S. cerevisiae, logarithmic growing, haploid cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: SK1 wild type haploid cells, Mating types and growth conditions: Mata and Matα cells were grown separately in YPD.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were flash frozen in liquid Nitrogen. Total RNA was extracted using RNeasy Midi kit (QIAGEN)
|
| Label |
Cy3
|
| Label protocol |
Samples and reference cDNA were synthesized from total RNA and labeled with Cy3 and Cy5 by the indirect amino-allyl method as described below. Total RNA (20 µg) was annealed with 4 µg oligo dT12-18 and reverse transcribed into cDNA with M-MLV Reverse Transcriptase RNase H Minus (Promega) for 2 hours at 45°C in the presence of 2.5mM MgCl2, 0.5 mM of dATP, dCTP and dGTP, 0.1mM dTTP, 0.4mM amino-allyl dUTP. Additional 400U M-MLV Reverse Transcriptase RNase H Minus was added and incubated for another 2 hours. The reaction was terminated by incubation at 70°C for 15 minutes and RNA was degraded by adding 10 µM 1N NaOH and incubation at 65 C for 30 minutes. The sample was neutralized by titrating with 1M HCl. cDNA was purified by ethanol precipitation, resuspended in 10 µl 0.1M Sodium Carbonate buffer pH 9.3. Cy5 or Cy3 were added to experimental or reference sample, respectively, and incubated at 25 C for 1 hour. Labeled cDNA was purified by Strataprep PCR purification kit (Stratagene) according to the manufactures instructions. Dye incorporation was measured using a spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
For each hybridization, labeled Cy3 and Cy5 were combined together with the following blockers: 5µg Herring sperm (Promega), 5 µg tRNA (Gibco) and 17.5µg Poly A (Synthesized oligos with the length of 40, 50 and 60), and concentrated to 25µl using Microcon (Millipore). 25 µl of hybridization x2 solution (10x SSC, 50% formamide and 0.2% SDS) was added. Microarrays containing all yeast ORFs (purchased from the Microarray Centre, University Health Network, Ontario) were pre-hybridized by incubating in a solution containing 1% BSA, 25% formamide, 5x SSC and 0.1% SDS at 42 C for 45 minutes. The slides were washed with DDW and dried by centrifugation (3 minutes, 2000 rpm). The labeled sample with the blockers, was boiled for 5 minutes, centrifuged for 1 minute and hybridized on the slide. The slides were placed in hybridization chamber (Corning) and incubated over night at 42 C. The slides were washed for 5 minutes at 42 C with a solution containing 2x SSC and 0.1% SDS. Additional wash was performed at room temperature with a solution containing 0.1x SSC and 0.1% SDS, followed by 3 additional washes at room temperature in 0.1x SSC solution.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using Aginlet's DNA microarray scanner and image intensity data were extracted and analyzed with SpotReader (Niles Scientific) analysis software.
|
| Description |
S. cerevisiae spores, 1 hour and 30 minutes after the transfer to nitrogen
|
| Data processing |
Background intensity was subtracted using a Bayesian correction, and the ratios of the two dyes were log2-transformed. Log2 ratios were then corrected for intensity-dependant and spatial-dependant biases by subtracting a Lowess curve followed by a median filter. The two spots corresponding to each gene were then averaged, and genes for which the two spots were significantly different were declared as 'missing values' (along with other genes which were flagged by the image analysis software or removed by manual inspection).
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| |
|
| Submission date |
Mar 24, 2007 |
| Last update date |
Dec 23, 2007 |
| Contact name |
Naama Barkai |
| E-mail(s) |
naama.barkai@weizmann.ac.il, simchen@vms.huji.ac.il
|
| Organization name |
The Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Lab |
Naama Barkai
|
| Street address |
P.O box 26
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL5031 |
| Series (2) |
| GSE7362 |
The contribution of different nutrients to spore germination in Saccharomyces cerevisiae |
| GSE7393 |
Spore germination in Saccharomyces cerevisiae |
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