Osmotic stress conditions were created by addition of KCl at 0.6 M. Experiments were initiated from exponential cultures growing in YPD. t0: before adding KCl and samples were taken at 8, 15, 30 and 45 minutes.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolated from yeast cells was prepared as described by Sherman et al. [1986], but using a multiple-sample automated device (Fast-Prep, BIO101) to break the cells. Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Spring Harbor Cold Laboratory Press, Cold Spring Harbor, New York.
Label
33P-UTP
Label protocol
In vivo labeling by run-on (GRO) was done using 33P-UTP. Around 6 x 10exp8 yeast cells were used to perform in vivo transcription. After spinning down cells, the cells were washed and then were permeabilized with 20 mL of 0.5% sarkosyl. In vivo transcription was performed by resuspending cells in 115 µl of RNA water and adding 162 µL of Run On Mix (120 µl of 2.5x transcription buffer [50 mM Tris-HCl pH 7.7, 500 mM KCl, 80 mM MgCl2], 20 µl of AGC mix [10 mM each of CTP, ATP and GTP], 6 µl of DTT [0.1 M] and 13 µl of [α-33P]UTP [3000 Ci/mmol, 10 Ci/µL]). The mix was incubated for 5 minutes at 30ºC to allow transcription elongation. The reaction was stopped by adding 1 ml of cold distilled water to the mix.
Hybridization protocol
Hybridization Solution was: 0.5M Na-Phosphate buffer, 1mM EDTA and 7% SDS, pH 7.2. The hybridization protocol used was as follows. macroarrays were inserted in 12.5X 2.5-cm flat-bottom plastic tubes and pre-hybridized in a rotator oven with 5 ml pre-hybridization solution (the same as used for hybridization but without the radioactive sample) at 65ºC. The pre-hybridization solution was then replaced with 3 ml of the same solution containing 1-5X10exp6 dpm/mL of radioactive sample and hybridized for 48h. Washing conditions were: 20 min at 65ºC in 1X SSC, 0.5% SDS, and twice at 65ºC for 10 min in 0.5X SSC, 0.1% SDS. After quantification, macroarrays were stripped by washing them once with 50 mM NaOH at 45ºC, once with 50 mM Tris-HCl, pH 7.5, 0.1x SSC, 0.1% SDS and once pouring with boiling stripping buffer (5 mM sodium phosphate, pH 7.5, 0.1% SDS) over the membrane and left to cool at room temperature. To ensure that radioactivity had been eliminated, the macroarrays were either checked with a Geiger counter.
Scan protocol
Images were acquired using a FujiFilm FLA3000 Phosphorimager. After the washing step, membranes were kept humid, sealed in Saran wrap, avoiding any bubbles, and exposed to an imaging plate (BAS-MP, FujiFilm) until quantification.
Description
Time 0 processed data: Run-on labelling for cbc1Δ strain gDNA hybridization: gDNA.v10-L1-F9.txt gDNA hybridization: gDNA.v10-L1-F10.txt gDNA hybridization: gDNA.v10-L3-F7.txt
Data processing
Raw data was pre-processed in Excel 2007 and comes from image quantization and local background subtracted. Only data above 10% regard to local background was considered. Each GRO and cDNA replicate were corrected by normalization with gDNA hybridization for corresponding microarray used (gDNA hybridization raw files are available in a tar archive on the series record). Processed data is the average that results from Median Absolute Desviation normalization of each gDNA corrected replicate using ArrayStat software. For RA (mRNA), average processed values were corrected by percentage of guanines and for TR (GRO) the average processed value for each gene was corrected by percentage of uridines present in each probe-coding strand. The mRNA levels was adjusted using the concentration of mRNA/cell for each strain regard the wild type strain determined by a dot blot assay and mRNA extractions as described in García-Martinez 2004.
Determination of transcriptional rates and mRNA levels during osmotic stress in S. cerevisiae wild type and cbc1/sto1 mutant cells using Genomic Run On (GRO)
Data table header descriptions
ID_REF
VALUE
Value is the median resulting from each replicate normalized by Median Absolute Deviation using ArrayStat software. Each replicate was corrected by gDNA hybridization from corresponding microarray. 'null' corresponds to Non Assigned value
