|
| Status |
Public on Jul 08, 2008 |
| Title |
yeast_on_acetate_N2_45min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
yeast on acetate, exposed 45 min to N2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
media: solid YP acetate
pre-grown overnight in liquid YPD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions were initiated by resuspending each pellet in 400 uL TES (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS) and 400 uL acid phenol (pre-warmed to 65 degrees) with vigorous vortexing. Cells were incubated at 65 degrees for 1 hour, with vortexing every 10 minutes. Suspensions were transferred to microfuge tubes and put on ice for 5 minutes, then centrifuged at top speed for 10 minutes at 4 degrees on an Eppendorf Minispin centrifuge. Aqueous phases were transferred to fresh tubes and extracted with 400 uL room temperature acid phenol twice more, followed by a single extraction with 400 uL chloroform. RNA was then precipitated from the aqueous phase using 40 uL 3M sodium acetate, pH 5.3 and 1 uL ice-cold ethanol, and centrifuged for 20 minutes. Pellets were washed with 0.5 mL ice-cold 80% ethanol and centrifuged for 5 minutes. RNA was redissolved in 20 uL water.
|
| Label |
Cy5
|
| Label protocol |
Diluted 4.0 ug aRNA in 4.5 uL water. Added 4.5 uL 0.2M sodium bicarbonate. Vortexed and collected by centrifugation. Added 5.5 uL of dye. Vortexed and collected by centrifugation. Incubated at room temperature for 1 hour in the dark.
|
| |
|
| Channel 2 |
| Source name |
yeast on acetate, exposed 45 min to air
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
media: solid YP acetate
pre-grown overnight in liquid YPD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions were initiated by resuspending each pellet in 400 uL TES (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS) and 400 uL acid phenol (pre-warmed to 65 degrees) with vigorous vortexing. Cells were incubated at 65 degrees for 1 hour, with vortexing every 10 minutes. Suspensions were transferred to microfuge tubes and put on ice for 5 minutes, then centrifuged at top speed for 10 minutes at 4 degrees on an Eppendorf Minispin centrifuge. Aqueous phases were transferred to fresh tubes and extracted with 400 uL room temperature acid phenol twice more, followed by a single extraction with 400 uL chloroform. RNA was then precipitated from the aqueous phase using 40 uL 3M sodium acetate, pH 5.3 and 1 uL ice-cold ethanol, and centrifuged for 20 minutes. Pellets were washed with 0.5 mL ice-cold 80% ethanol and centrifuged for 5 minutes. RNA was redissolved in 20 uL water.
|
| Label |
Cy3
|
| Label protocol |
Diluted 4.0 ug aRNA in 4.5 uL water. Added 4.5 uL 0.2M sodium bicarbonate. Vortexed and collected by centrifugation. Added 5.5 uL of dye. Vortexed and collected by centrifugation. Incubated at room temperature for 1 hour in the dark.
|
| |
|
| |
| Hybridization protocol |
Dissolved 15g powdered non-fat milk in 370mL water, 120mL 20x SSC, 5mL 10% SDS. Warmed solution in 42 degree water bath for ~45 minutes. Put slides into solution and incubated for 1 hour at 42 degrees. Washed slides vigorously in water, then in 100% 2-propanol. Dried slides by spinning at 500xg for 5 minutes. aRNA samples were thawed. 10% SDS and 20x SSC were added to attain final concentrations of 0.2% SDS in 3x SSC. Heated samples at 99 degrees for 5 minutes. Recollected samples by centrifugation. Added sample onto array area of slides and covered with coverslip. Incubated slides ~16 hours in sealed hybridization chamber at 63 degrees. After hybridization, washed slides in 1x SSC/ 0.03% SDS. Coverslips were separated from slides by gentle agitation during washing. Then washed slides in 1x SSC. Soaked slides in 0.2x SSC while shaking at 60 RPM in the dark for 20 minutes. Soaked slides in 0.05x SSC while shaking at 60 RPM in the dark for 20 minutes. Dried slides by gentle centrifugation for 5 minutes.
|
| Scan protocol |
Arrays were scanned using an Axon Instruments GenePix4000B scanner at 635 and 532 nm with 10 micron resolution. PMT settings for wavelengths were adjusted and balanced using GenePix Pro 6.0 software.
|
| Description |
This array is one of a series of arrays of yeast whose cell divisions are reversibly arrested when made anoxic on solid acetate media, using either pure nitrogen or pure carbon monoxide.
|
| Data processing |
Image files were loaded onto GenePix Pro 6.0 software. Auto-alignment was applied. Bad spots were flagged. Locations of misaligned spots were corrected. Images were then analyzed to generate .gpr files. .gpr files were filtered using GDFilter, a program developed at FHCRC to remove data from spots < 75 microns in diameter or signal-to-noise ratios < 3. Data were loaded onto a GeneTraffic server. The “Lowess (Sub-Grid, non-flagged)” normalization was applied. Data was then exported using GTExport, another application developed at FHCRC.
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| |
|
| Submission date |
Jul 03, 2008 |
| Last update date |
Jul 07, 2008 |
| Contact name |
Kin Chan |
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL1914 |
| Series (1) |
| GSE12004 |
Transcriptional profiles of yeast on acetate exposed to pure nitrogen or carbon monoxide |
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