Yeast strain S288C was grown until the exponential phase (A660, 1.0) in 600 ml YPD medium in a 3 L Erlenmeyer flask at 30 °C. An aliquot of 400 ml in a 3 L Erlenmeyer flask was frozen at −80 C without the addition of a cryoprotectant, and stored for 1 week. The frozen cell culture was then thawed at 30 °C in a water-bath for 25 min. The thawed cells were further incubated and an aliquot of 100 ml was sampled from the culture at 15 min after complete thawing.
Extracted molecule
polyA RNA
Extraction protocol
Hot-phenol and oligotex dt mRNA purification kit (TaKaRa)
Label
Cy5-dUTP
Label protocol
At least 4 µg of mRNA and a 0.4 µM oligo dT12-17 primer (GIBCO BRL; Life Technologies) were denatured at 70 ºC for 5 min, and the labeled cDNA was synthesized. The reaction mixture contained 0.5 mM each of dATP, dGTP, and dCTP; 0.2 mM dTTP (GIBCO BRL; Life Technologies); 15 mM Cy5-labeled dUTP (PE Applied Biosystems); 200 units of PowerScript Reverse Transcriptase (CLONTEC, Palo Alto, CA); and an appropriate buffer and RNase inhibiter (GIBCO BRL; Life Technologies). The reactions were performed at 42 ºC for 1h and 30 min. Another 200 units of transcriptase was added in the tubes 40 min after the beginning of reverse transcription. The template mRNA was dissolved by the addition of 0.5 N NaOH for 1 h at 65 ºC. After neutralization with 1 M Tris-HCl, Cy3- and Cy5-labeled cDNA solution was mixed and filtered on Microcon 30 (AMICON).
Yeast strain S288C was grown until the exponential phase (A660, 1.0) in 600 ml YPD medium in a 3 L Erlenmeyer flask at 30 °C. An aliquot of 200 ml was taken from the cell culture and used as the control without freezing.
Extracted molecule
polyA RNA
Extraction protocol
Hot-phenol and oligotex dt mRNA purification kit (TaKaRa)
Label
Cy3-dUTP
Label protocol
At least 4 µg of mRNA and a 0.4 µM oligo dT12-17 primer (GIBCO BRL; Life Technologies) were denatured at 70 ºC for 5 min, and the labeled cDNA was synthesized. The reaction mixture contained 0.5 mM each of dATP, dGTP, and dCTP; 0.2 mM dTTP (GIBCO BRL; Life Technologies); 15 mM Cy3-labeled dUTP (PE Applied Biosystems); 200 units of PowerScript Reverse Transcriptase (CLONTEC, Palo Alto, CA); and an appropriate buffer and RNase inhibiter (GIBCO BRL; Life Technologies). The reactions were performed at 42 ºC for 1h and 30 min. Another 200 units of transcriptase was added in the tubes 40 min after the beginning of reverse transcription. The template mRNA was dissolved by the addition of 0.5 N NaOH for 1 h at 65 ºC. After neutralization with 1 M Tris-HCl, Cy3- and Cy5-labeled cDNA solution was mixed and filtered on Microcon 30 (AMICON).
Hybridization protocol
Hybridized for 16-18 h at 65 C
Scan protocol
Scan Array 4000 (PerkinElmer Japan Co., Ltd) was used for scanning. Array images were analyzed with Gene Pix Pro 4.0 (Axon Instrument).
Description
Data with flag value 0 were used for LOWESS normalization processing.
Cy5 intensity surrounding each spot (background), (median)
Cy3F
Cy3 inner intensity (median)
Cy3B
Cy3 intensity surrounding each spot (background), (median)
Cy5FB
Cy5 intensity subtracted from background intensity
Cy3FB
Cy3 intensity subtracted from background intensity
FlagGPR
Flag from GenePix Pro analysis
Flag
Additional flags by LOCUS_NAME1 as following; TE, TE; NG, NGT; QU, _ or -; C3, Cy3; C5, Cy5; EM, empty. Additional flags by Cy5F and Cy3F as following; BL, both data Cy3F and Cy5F is less than reliable limit; 5L, Cy5F is less than reliable limit; 3L, Cy3F is less than reliable limit; BM, both data Cy3F and Cy5F is over than scanning range; 5M, Cy5F is over than scanning range; 3M, Cy3F is over than scanning range.
