|
| Status |
Public on Apr 30, 2015 |
| Title |
SDP8 + 24h DOX Exp_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
SDP8 conditional mutant strain treated with doxycycline for 24 h
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: teto7: PKH2 pkh1::HIS3 pkh3::NatMX4
treatment: with doxycycline for 24 h
strain: CML476
|
| Treatment protocol |
Treated with 100 microg/ml doxycycline, from a 5 g/l stock in an ethanol:water mix (1:1), for 24h.
|
| Growth protocol |
Saturated culture grown in YPD medium was diluted at OD660 0.05 in YPD supplemented with doxycycline. Eight h later, when OD660: 0.609, a new culture was prepared at initial OD660: 0.01 that was also supplemented with doxycycline and was grown for 16 h. Cells present in 50 ml of the culture were collected by centrifugation and washed with cold water. The dried cell pellet was kept at -80 ºC until RNA purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA purification using the Ambion RiboPure™-Yeast Total RNA Isolation Kit.
|
| Label |
Cy3
|
| Label protocol |
Fluorescent labeled cDNA was prepared from 8 microg of purified total RNA by the indirect dUTP-labeling method using the CyScribe post-labeling kit (GE Healthcare) and oligo(dT) as a primer.
|
| |
|
| Channel 2 |
| Source name |
Wild type strain treated with doxycycline for 24 h
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: CML476
treatment: with doxycycline for 24 h
|
| Treatment protocol |
Treated with 100 microg/ml doxycycline, from a 5 g/l stock in an ethanol:water mix (1:1), for 24h.
|
| Growth protocol |
Saturated culture grown in YPD medium was diluted at OD660 0.05 in YPD supplemented with doxycycline. Eight h later, when OD660: 0.554, a new culture was prepared at initial OD660: 0.01 that was also supplemented with doxycycline and was grown for 16 h. Cells present in 50 ml of the culture were collected by centrifugation and washed with cold water. The dried cell pellet was kept at -80 ºC until RNA purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA purification using the Ambion RiboPure™-Yeast Total RNA Isolation Kit.
|
| Label |
Cy5
|
| Label protocol |
Fluorescent labeled cDNA was prepared from 8 microg of purified total RNA by the indirect dUTP-labeling method using the CyScribe post-labeling kit (GE Healthcare) and oligo(dT) as a primer.
|
| |
|
| |
| Hybridization protocol |
Prehybridizations of the DNA microarrays were carried out at 42 ºC for 1 h in a solution containing 5x SSC, 0.1% SDS, 1% bovine serum albumin. For hybridization, dried Cy3-and Cy5-labelled probes were resuspended in 35 ul of hybridization solution (50% formamide, 5x SSC, 0.1% SDS) each and mixed. Five ug of salmon sperm DNA was added to the mix before denaturation for 3 min at 95 ºC. DNA microarrays were hybridized in an ArrayBooster hybridization station (Sunergia Group) for 14 h at 42 ºC.
|
| Scan protocol |
The scanner ScanArray 4000 (Packard) was used to obtain the Cy3 and Cy5 images with a resolution of 10 micro m.
The fluorescent intensity of the spots was measured and processed using the GenePix Pro 6.0 software (Molecular Devices)
|
| Description |
The strain SDP8 (CML476 background), contains a KanMX4-tetO7 cassette replacing about 0.5 kbp of the PKH2 promoter, and located immediately before the PKH2 coding region.
Spots with either a diameter smaller than 120 um, or fluorescence intensity for Cy3 and Cy5 lower than 100 units, were not considered for further analysis.
|
| Data processing |
The Gene Expression Profile Analysis Suite, GEPAS v4.0 package (http://www.gepas.org/) was used to the processing of data: the Preprocessing tool was used to merge replicates and give the average value.
The normalization method we used to generate the data reported in the VALUE columns was the ratio of medians for the non-flagged genes. Spots with saturated values were excluded from the normalization process.
|
| |
|
| Submission date |
Oct 05, 2011 |
| Last update date |
Apr 30, 2015 |
| Contact name |
Antonio Casamayor |
| E-mail(s) |
antonio.casamayor@uab.es
|
| Organization name |
Universitat Autonoma de Barcelona
|
| Lab |
Biol. Mol. Llevat
|
| Street address |
Edifici IBB, Campus UAB
|
| City |
Cerdanyola |
| State/province |
Barcelona |
| ZIP/Postal code |
08193 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10039 |
| Series (1) |
| GSE32623 |
Depletion of yeast PDK1 orthologs triggers a stress-like transcriptional response |
|
