Yeast strains were collected from glycerol stocks stored at -80°C with a hot sterile rod, without letting the stock to thaw. These cells were grown o/n and used to inoculate fresh synthetic complete medium at a density of about 2*10^5 cell/ml. Cells were then collected during exponential growth (beteween) 3*10^6 e 7*10^6 and used for RNA extraction as described in extract protocol.
Extracted molecule
total RNA
Extraction protocol
Typically 1*10^9 cells/sample were collected by filtration, washed two times with cold water and immediately freezed at –80°C. For RNA extraction, samples were kept on ice till they were completely thawed. Cells were resuspended in LETS 2X buffer (0.2M LiCl, 0.02M EDTA, 0.4% SDS, 0.02M Tris-HCl, pH=7.4) and phenol/chloroform/isoamylalcohol (PCI) (25:24:1, v/v). After the addition of acid-washed glass beads (Sigma), cells were broken by three vortex/ice cycles of 45 sec each on ice. Suspension was centrifuged (30’, 13000 rpm) and the upper phase was extracted twice with about one volume of PCI. The acqueous layer was collected and precipitated with LiCl (0.5M final concentration) and ethanol at -80°C. Total RNA was purified using the RNeasy RNA purification kit (Quiagen) and stored in RNase-free water.
Label
biotin
Label protocol
cRNA was generated by using the Affymetrix One-Cycle Target Labeling and Control Reagent kit (Affymetrix Inc., Santa Clara, CA) following the protocol of the manufacturer.
Hybridization protocol
cRNA was generated by using the Affymetrix One-Cycle Target Labeling and Control Reagent kit (Affymetrix Inc., Santa Clara, California, USA), following the manufacturer’s protocol. The biotinylated cRNA was hybridized to the YG_S98 Affymetrix DNA chips, containing 9335 probe sets which represent approximately 7000 genes and open reading frames from S. cerevisiae Genome database. Chips were washed and scanned on the Affymetrix Complete GeneChip Instrument System, generating digitized image data (DAT) files.
Scan protocol
Chips were washed and scanned on the Affymetrix Complete GeneChip Instrument System, generating digitized image data files.
Coordinated increase in cellular RNA and protein content induced by overexpression of Far1, a cyclin dependent kinase inhibitor, involves large transcriptional reprogramming and requires the Sfp1 protein.
