In vivo labeled total RNA from W303-1a yeast strain. t41: 41 minutes after adding terbutyl. Hybridization on macrochip F5.
Treatment protocol
Oxidative stress conditions were created by addition of t-butyl hydroperoxide (t-BOOH) at 0.1 mM. Experiments were initiated on exponential cultures that had been grown in such conditions for at least ten generations, at concentrations of 1.5-2 x 10exp7 cells/ml. t41: 41 minutes after adding t-butyl.
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) using 33P-UTP. Around 6 x 10exp8 yeast cells were used to perform in vivo transcription. After spinning down cells, they were washed in cold water and the cell pellet was resuspended in 900 µl of sterile cold water (final volume 950 µl). Then, the cell suspension was transferred to a fresh micro centrifuge tube, 50 µl of 10% sarkosyl were added and cells were incubated for 20 minutes on ice. After the permeabilization step, cells were recovered by low-speed centrifugation and the supernatant was removed. In vivo transcription was performed by resuspending cells in 120 µl of 2.5x transcription buffer (50 mM Tris-HCl pH 7.7, 500 mM KCl, 80 mM MgCl2), 16 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 μCi/µL). Cells were maintained on ice at all times. The final volume was adjusted to 300 µl with distilled water and 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: 5XSSC, 5XDenhart’s, 0.5% SDS and 100 µg herring sperm DNA/ml. The hybridization protocol used was as follows. Filters were inserted in 12.5X2.5-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 3X10exp6 dpm/mL of radioactive sample and hybridized for 44h. Washing conditions were: 1h at 65ºC for 20 min in 2XSSC, 0.1% SDS, and twice at 65ºC for 30 min in 0.2XSSC, 0.1% SDS.
Scan protocol
Images were acquired using a FujiFilm FLA3000 Phosphorimager.
Description
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 various times. Filters 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 filters were either checked with a Geiger counter or re-scanned with the Phosphorimager.
