strain: W303 background: SSD1-V time point: 10 min
Treatment protocol
Cells were arrested with alpha factor, and released into YEPD to get a synchronized population. Cells were sampled every 10 min after release.
Growth protocol
Cells were grown in YEPD medium to early log phase before arrest by alpha factor. For the yeast asynchronous population control, cells were grown overnight to an OD of 0.6 in YEPD before harvest.
Extracted molecule
total RNA
Extraction protocol
RNA extractions initiated by spinning down and wash cell pellet with 1 ml cold RNA buffer. Freeze cell pellet in dry ice, then thaw cells, resuspend in 100-120 µl ice cold RNA-buffer. Add 1/2 volume of acid washed glass beads, vortex at highest level 3 min in cold room. Add 450 µl RNA-buffer-SDS (RT), vortex briefly, add 450 µl equilibrated Phenol, vortex at highest level 3 min in cold room. Spin full speed 10 min, transfer upper phase to fresh tube, add 300 µl equilibrated Phenol, vortex, add 300 µl Chloroform, vortex, spin 2 min, extract upper phase once again with Chloroform. Add 20 µl 4 M NaCl, 1 ml Ethanol, precipitate 30 min at -20 to -80 °C. Spin full speed 10 min, wash pellet with 150 µl 70 % Ethanol, air dry pellet, resuspend in 30-50 µl H2O.
Label
Cy5
Label protocol
Thirty micrograms of total RNA was used for cDNA synthesis. The cDNA was coupled to Cy5 using an amino-allyl dye-coupling procedure (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). RNA from an asynchronous population of W303a cells was used as a control, and its cDNA was labeled with Cy3 dye.
Cells were grown in YEPD medium to early log phase before arrest by alpha factor. For the yeast asynchronous population control, cells were grown overnight to an OD of 0.6 in YEPD before harvest.
Extracted molecule
total RNA
Extraction protocol
RNA extractions initiated by spinning down and wash cell pellet with 1 ml cold RNA buffer. Freeze cell pellet in dry ice, then thaw cells, resuspend in 100-120 µl ice cold RNA-buffer. Add 1/2 volume of acid washed glass beads, vortex at highest level 3 min in cold room. Add 450 µl RNA-buffer-SDS (RT), vortex briefly, add 450 µl equilibrated Phenol, vortex at highest level 3 min in cold room. Spin full speed 10 min, transfer upper phase to fresh tube, add 300 µl equilibrated Phenol, vortex, add 300 µl Chloroform, vortex, spin 2 min, extract upper phase once again with Chloroform. Add 20 µl 4 M NaCl, 1 ml Ethanol, precipitate 30 min at -20 to -80 °C. Spin full speed 10 min, wash pellet with 150 µl 70 % Ethanol, air dry pellet, resuspend in 30-50 µl H2O.
Label
Cy3
Label protocol
Thirty micrograms of total RNA was used for cDNA synthesis. The cDNA was coupled to Cy5 using an amino-allyl dye-coupling procedure (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). RNA from an asynchronous population of W303a cells was used as a control, and its cDNA was labeled with Cy3 dye.
Hybridization protocol
Cy5-labeled cDNA from each timepoint was mixed with Cy3-labeled control cDNA, and hybridized to yeast cDNA microarrays
Scan protocol
Arrays were scanned using an Axon Instruments GenePix4000B scanner, and image analysis was performed using GenePix Pro microarray acquisition and analysis software.
Description
Transcript profiling of yeast W303 SSD1-V cell cycle.
Data processing
LOWESS normalized using agilent software (Genespring 7).
