|
| Status |
Public on Nov 07, 2014 |
| Title |
rcm1ko_polysomes_90min_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
exponentially growing yeast cells (OD = 0.6)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: rcm1Δ
h2o2 stress [min]: 90
assayed molecule: polysomal RNA
|
| Treatment protocol |
After the culture reached an OF of 0.5 - 0.6, 0.4 mM H2O2 were added to the medium for 30 min or 90 min.
|
| Growth protocol |
Wild-type and rcm1 knockout yeast were grown overnight in 3 mL SC medium at 28°C with shaking. On the next day, 100 ml SD were inoculated with the overnight culture to an OD600 of 0.003 and grown at 28°C with shaking until the OD600 reached 0.5 - 0.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
125 µg/ml cycloheximide (Sigma) were added and the cells were incubated at 28°C with shaking for further 15 min. Afterwards the flasks were placed on ice for 15 min with shaking from time to time. After pelleting the cells at 3,500 rpm and 4°C for 5 min, the pellets were washed two times with 2 mL cooled TMNSH buffer (10 mM Tris pH 7.4, 50 mM NH4Cl, 10 mM MgCl2, 12.5 mM 2-mercaptoethanol) and resuspended in 1 mL of lysis buffer (10 mM Tris pH 7.4, 50 mM NH4Cl, 10 mM MgCl2, 12.5 mM 2-mercaptoethanol, 50 mM KCl, 1 mM PMSF, 125 μg/ml cycloheximid). Lysis was performed by transferring the suspensions to glass tubes and vortexing with glass beads (0.4 - 0.6 mm diameter) 8 times for 30 sec, with at least 30 sec recovery on ice between each cycle. Afterwards the crude lysates were centrifuged at 4,000 rpm at 4°C for 10 min to remove insoluble debris. The supernatants were transferred to fresh tubes and were supplemented with 100 μl Triton X-100 (10% v/v). After centrifugation at full speed in a tabletop centrifuge at 4°C for 10 min, the supernatants were carefully transferred to fresh vials and the OD260 was recorded for quantification. A volume equalling 15 OD260 was loaded onto a 10 mL linear 7-52 % sucrose gradient in TMNSH buffer containing 50 mM KCl and centrifuged at 37,000 rpm at 4°C for 3 h. The profiles were recorded from the bottom of the tubes with a UV detector set to 254 nm. 300 µl polysomal fractions or total lysate were mixed with 900 µl Trizol LS and RNA was extracted following the manufacture's protocol.
|
| Label |
CY3
|
| Label protocol |
Samples were labelled using the Low Input Quick Amp Labeling Kit Two Color (Agilent) following the manufactures instructions.
|
| |
|
| Channel 2 |
| Source name |
exponentially growing yeast cells (OD = 0.6)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: pool of wild-type and rcm1Δ
h2o2 stress [min]: pool of 0, 30 and 90 min
assayed molecule: pool of polysomal RNA and total RNA
|
| Treatment protocol |
After the culture reached an OF of 0.5 - 0.6, 0.4 mM H2O2 were added to the medium for 30 min or 90 min.
|
| Growth protocol |
Wild-type and rcm1 knockout yeast were grown overnight in 3 mL SC medium at 28°C with shaking. On the next day, 100 ml SD were inoculated with the overnight culture to an OD600 of 0.003 and grown at 28°C with shaking until the OD600 reached 0.5 - 0.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
125 µg/ml cycloheximide (Sigma) were added and the cells were incubated at 28°C with shaking for further 15 min. Afterwards the flasks were placed on ice for 15 min with shaking from time to time. After pelleting the cells at 3,500 rpm and 4°C for 5 min, the pellets were washed two times with 2 mL cooled TMNSH buffer (10 mM Tris pH 7.4, 50 mM NH4Cl, 10 mM MgCl2, 12.5 mM 2-mercaptoethanol) and resuspended in 1 mL of lysis buffer (10 mM Tris pH 7.4, 50 mM NH4Cl, 10 mM MgCl2, 12.5 mM 2-mercaptoethanol, 50 mM KCl, 1 mM PMSF, 125 μg/ml cycloheximid). Lysis was performed by transferring the suspensions to glass tubes and vortexing with glass beads (0.4 - 0.6 mm diameter) 8 times for 30 sec, with at least 30 sec recovery on ice between each cycle. Afterwards the crude lysates were centrifuged at 4,000 rpm at 4°C for 10 min to remove insoluble debris. The supernatants were transferred to fresh tubes and were supplemented with 100 μl Triton X-100 (10% v/v). After centrifugation at full speed in a tabletop centrifuge at 4°C for 10 min, the supernatants were carefully transferred to fresh vials and the OD260 was recorded for quantification. A volume equalling 15 OD260 was loaded onto a 10 mL linear 7-52 % sucrose gradient in TMNSH buffer containing 50 mM KCl and centrifuged at 37,000 rpm at 4°C for 3 h. The profiles were recorded from the bottom of the tubes with a UV detector set to 254 nm. 300 µl polysomal fractions or total lysate were mixed with 900 µl Trizol LS and RNA was extracted following the manufacture's protocol.
|
| Label |
CY5
|
| Label protocol |
Samples were labelled using the Low Input Quick Amp Labeling Kit Two Color (Agilent) following the manufactures instructions.
|
| |
|
| |
| Hybridization protocol |
Hybridisation to Yeast (V2) 8 x 15K Gene Expression Microarrays (Agilent) and washing were performed following the manufactures instructions.
|
| Scan protocol |
Slides were scanned with the two laser Agilent microarray scanner (G2565A). For raw data generation Agilent feature extraction software (version 7.5) was used.
|
| Description |
Polysome-associated RNA fraction of 90 min H2O2 treated rcm1 knockout yeast (CY3) vs. common reference pool (CY5)
|
| Data processing |
Raw data were processed into R/Bioconductor. Intensities were log2 transformed, backgrounds were subtracted using normexp, and intensities were normalized within arrays by using the Loess method and between arrays by using Aquantile.
|
| |
|
| Submission date |
Nov 06, 2014 |
| Last update date |
Nov 07, 2014 |
| Contact name |
Markus Schosserer |
| E-mail(s) |
markus.schosserer@meduniwien.ac.at
|
| Organization name |
Medical University of Vienna
|
| Department |
Center for Pathobiochemistry and Genetics
|
| Lab |
Markus Schosserer
|
| Street address |
Waehringer Strasse 10
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE63030 |
Yeast translational profiling: rcm1 knockout vs. wild-type with oxidative stress |
|
