|
| Status |
Public on Sep 30, 2022 |
| Title |
Raw TR data S454P mutant replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
S454P mutant
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Y841
genotype: S454P mutant
method: GRO
|
| Growth protocol |
Cells were grown in YPD at 30ºC for at least 15 hours to reach early exponential phase at 0.4-0.5 OD600. Then, cells were spun-down and collected. Each sample was done in triplicate.
|
| 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 |
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 |
GRO labelling for S454P strain, Replicate 2
|
| Data processing |
Raw image quantization with background subtracted and Median Absolute Desviation normalization of each triplicate. The average processed value for each gene was corrected by percentage of guanines (RA data) or uracils (TR data) present in each probe-coding strand as described in García-Martinez et al. 2004.
Value in the processed data is the result of the Median Absolute Desviation normalization of each triplicate of the raw data.
|
| |
|
| Submission date |
Sep 20, 2020 |
| Last update date |
Sep 30, 2022 |
| Contact name |
Jose E. Perez-Ortin |
| E-mail(s) |
jose.e.perez@uv.es
|
| Phone |
34 963 543467
|
| Organization name |
Universitat de Valencia
|
| Department |
Bioquimica y Biologia Molecular
|
| Lab |
Yeast Functional Genomics (GFL)
|
| Street address |
Dr. Moliner 50
|
| City |
Burjassot |
| State/province |
Valencia |
| ZIP/Postal code |
E46100 |
| Country |
Spain |
| |
|
| Platform ID |
GPL24366 |
| Series (1) |
| GSE158250 |
Transcriptomic analysis of mutant yeast strains deficient in Xrn1 nuclear import |
|
