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| Status |
Public on Sep 15, 2017 |
| Title |
Taf5 anchor away, plus rapamycin, newly-synthesized RNA, replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
S. cerevisiae culture in exponential growth
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
parental strain: W303
genotype: S. cerevisiae: MATalpha tor1-1; fpr1::NAT; RPL13A-2xFKB12::TRP1; TAF4-FRB::HYGRO
molecule: Newly-synthesized RNA
treatment: after exposure to rapamycin
|
| Treatment protocol |
The culture was divided into two equal volumes of 100mL each and to one of those cultures rapamycin was added (+RAPA) until a final concentration of 1μg/mL, for 30 min, to allow nuclear depletion of either Taf4 or Taf5. To the other half of the culture, the control samples (-RAPA/minus rapamycin), only the vehicle (DMSO) was added. Newly synthesized RNAs were labeled for 6 min by adding 4-thiouracil (Sigma-Aldrich) until a final concentration of 5mM. In parallel, wild-type S. pombe cells were similarly grown in YES medium, at 31°C, and labeled for 6 min to be used as a spike-in across all samples. Cells were immediately pelleted and flash-frozen in liquid N2 and stored at -80°C until further use. Immediately before freezing the cells, a small aliquot was collected for cell counting purposes. S. cerevisiae and S. pombe cells were mixed in a ratio of 3:1.
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| Growth protocol |
For each yeast strain and condition, 200mL of S. cerevisiae cells were grown in YPD medium at 30°C to an OD600 ≈ 0.8.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using RiboPure yeast kit (Ambion, Life Technologies) according to the description provided by the manufacturer. This first sample is the steady-state RNA fraction. 200μg of total RNA was biotinylated using 200μL of 1mg/mL EZ-link HPDP- Biotin (Pierce) in 100μL of biotinylation buffer (100mM Tris- HCl at pH 7.5, 10mM EDTA) and adjusted to a final volume of 1000μL with DEPC-treated RNase-free water (Sigma-Aldrich) for 3 h at room temperature, protected from light and gentle agitation. After chloroform extraction and isopropanol precipitation, newly-synthesized biotinylated RNAs were bound to 100μL of μMACS streptavidin microbeads. Purification of labeled RNA was then carried out using μMACS streptavidin starting kit (Miltenyi Biotec). Labeled RNAs were eluted twice with 200μL of 100mM DTT and precipitated overnight. This purified labeled RNA is the newly-synthesized RNA used for the array hybridization.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA targets were prepared from 150ng of total RNA using the “MessageAmp™ Premier RNA Amplification Kit from Ambion, according to the Instruction Manual # 4386269 Revision B, 18 september 2007.
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| |
|
| Hybridization protocol |
Following fragmentation, 4μg of cRNAs were hybridized for 16 hours at 45°C, 60 rpm on GeneChip® Yeast Genome 2.0 arrays (Affymetrix).
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| Scan protocol |
The chips were washed and stained in the GeneChip® Fluidics Station 450 (Affymetrix) using the FS450_0003 script and scanned with the GeneChip® Scanner 3000 7G (Affymetrix) at a resolution of 1.56µm.
|
| Description |
Gene expression nascent RNA in a Taf5 anchor-away strain after exposure to rapamycin
|
| Data processing |
Raw data (.CEL Intensity files) were extracted from the scanned images using the Affymetrix GeneChip® Command Console (AGCC) version 4.1.2. CEL files were further processed with Affymetrix Expression Console software version 1.4.1 to calculate probeset signal intensities, using the Affymetrix statistics-based algorithms MAS-5.0 with default settings and global scaling as normalization method. The trimmed mean target intensity of each chip was arbitrarily set to 100.
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| |
|
| Submission date |
Mar 20, 2017 |
| Last update date |
Sep 17, 2017 |
| Contact name |
Didier Devys |
| E-mail(s) |
devys@igbmc.fr
|
| Organization name |
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
|
| Street address |
1 rue Laurent Fries / BP 10142
|
| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE96830 |
Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID |
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