Cells were grown in YPD at 30ºC for at least 15 hours to reach early exponential phase at 0.4-0.5 OD600. Then, an aliquot of 50 mL of cells were spun-down and collected for t0 sample. Auxin was added to a final concentration of 0.5 mM and two addtional aliquots were taken after 20 and 60 minutes. The experiment was done in duplicate.
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-dCTP
Label protocol
For GRO samples: 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, they were frozen in liquid nitrogen. Samples were then permeabilized with 1 mL of 0.5% sarkosyl and cells were recovered by low-speed centrifugation and the supernatant was removed. 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. For mRNA samples: cDNA was synthetized by reverse transcription of total mRNA using oligo d(T)15VN as primer and 33P-dCTP for labelling.
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. Filters 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 5 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, filters were stripped by washing them by 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 filters 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) for variable times.
Description
cDNA labelling for t60 sample. Replicates 1-2
Data processing
Raw image quantization with background subtracted for each of the two samples and averaged. The average processed value for each gene was corrected by percentage of guanines (RA data) or uracils (SR data) present in each probe-coding strand as described in García-Martinez et al. 2004.
