|
| Status |
Public on Jun 05, 2017 |
| Title |
GRO (1) spt16-140 TEToff::ARB1 strain 26ºC 4h after Doxicyline |
| Sample type |
RNA |
| |
|
| Source name |
spt6-140 TEToff::ARB1 at 26ºC
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/genotype: TEToff::ARB1 spt6-140 BY4741 background
|
| Growth protocol |
Yeast strains grown in YPD al 26ºC to exponential phase and treated with 5 µg/mL Doxicycline for 4 or 8h before GRO or RPCC (RNA pol II Chromatin Immunoprecipitation-Chip hybridization)
|
| 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. Total DNA was extracted from cells or from immunopreciptates as described in Pelechano et al. (2009) Plos Genet. e1000614.
|
| 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, they were permeabilized with 20 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. dCTP labelling of DNA was done by random primming as described in García-Martínez et al. (2004) Mol. Cell 15:303-13.
|
| |
|
| 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 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.
|
| 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 various times.
|
| Description |
First replica: Run-on labelling for spt6-140 strain on filter V8-L1-9
|
| Data processing |
Raw image quantization with background subtracted and Median Absolute Desviation normalization of each duplicate. Elaborated data for GRO were obtained by dividing each individual sample by genomic DNA signal from the same filter. For RPCC IP data were divided by WCE data obtained from the same sample and hybridized on the same filter. For GRO the value for each gene was corrected by percentage of uridines present in each probe-coding strand. For RPCC and gDNA value for each gene was corrected by percentage of G+C present in each probe.
Value was the result of the local background subtraction to the raw data. In Processed data, we normalized using ArrayStat software (Median Absolute Deviation).
|
| |
|
| Submission date |
Jan 13, 2015 |
| Last update date |
Jun 06, 2017 |
| Contact name |
Jose E. Perez-Ortin |
| E-mail(s) |
[email protected]
|
| 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 |
GPL8568 |
| Series (1) |
| GSE64921 |
Determination of the nascent transcriptional rates in a TEToff::ARB1 conditional mutant. |
|
