|
| Status |
Public on Nov 29, 2011 |
| Title |
Mut vs. WT biological repeat1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mutant delta-PrtT A
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
strain: mutant delta-PrtT A
|
| Treatment protocol |
growth on skim-milk medium of mutant and wild-type strains and subsequent analysis of transcriptional profiles
|
| Growth protocol |
1x107 wild type (Af293 strain) and mutant delta-PrtT A. fumigatus conidia were grown in 50 ml of 1% skim milk medium for 24 h at 37°C in an orbital incubator at 180 rpm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mycelium (wild type or delta PrtT) was subsequently freeze-dried. Total RNA from three independent biological repeats was extracted. Total RNA was isolated from each strain using the Qiagen RNeasy plant kit (Qiagen, Valencia, CA) following the filamentous fungus protocol.
|
| Label |
Cy3,Cy5
|
| Label protocol |
cDNA was generated from 2 µg of total RNA by incubating for 18 h at 42°C with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), a dNTP mixture containing amino-allyl dTTP and primed with random hexamers. Unincorporated dNTPs and hexamers were removed by purification with a Qiagen PCR purification column. Samples were then incubated for 18 h at room temperature with either Cy-3 or Cy-5 dye. After labeling, unincorporated dye was removed by purification with a Qiagen PCR purification column. Dye-coupled cDNA probes were evaporated and resuspended in hybridization buffer (40% formamide, 5X SSC, 0.1% SDS, 0.1 mM DTT and 6% salmon sperm DNA). The probe was heated for two cycles of 5 min at 95°C and then applied to the slide
|
| |
|
| Channel 2 |
| Source name |
wild type (Af293 strain)
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
strain: wild type Af293
|
| Treatment protocol |
growth on skim-milk medium of mutant and wild-type strains and subsequent analysis of transcriptional profiles
|
| Growth protocol |
1x107 wild type (Af293 strain) and mutant delta-PrtT A. fumigatus conidia were grown in 50 ml of 1% skim milk medium for 24 h at 37°C in an orbital incubator at 180 rpm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mycelium (wild type or delta PrtT) was subsequently freeze-dried. Total RNA from three independent biological repeats was extracted. Total RNA was isolated from each strain using the Qiagen RNeasy plant kit (Qiagen, Valencia, CA) following the filamentous fungus protocol.
|
| Label |
Cy3,Cy5
|
| Label protocol |
cDNA was generated from 2 µg of total RNA by incubating for 18 h at 42°C with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), a dNTP mixture containing amino-allyl dTTP and primed with random hexamers. Unincorporated dNTPs and hexamers were removed by purification with a Qiagen PCR purification column. Samples were then incubated for 18 h at room temperature with either Cy-3 or Cy-5 dye. After labeling, unincorporated dye was removed by purification with a Qiagen PCR purification column. Dye-coupled cDNA probes were evaporated and resuspended in hybridization buffer (40% formamide, 5X SSC, 0.1% SDS, 0.1 mM DTT and 6% salmon sperm DNA). The probe was heated for two cycles of 5 min at 95°C and then applied to the slide
|
| |
|
| |
| Hybridization protocol |
The probe was heated for two cycles of 5 min at 95°C and then applied to the slide and incubated at 42°C for 18 h. After post-incubation washes (two cycles of 55°C in 2X SSC, 0.1% SDS; room temperature (RT) in 0.1X SSC, 0.1% SDS and RT 0.1X SSC)
|
| Scan protocol |
slides were scanned with a Genepix 6000B scanner (Molecular Devices LLC, Sunnyvale, CA) to generate images that were then analyzed with the Spotfinder program
|
| Description |
Biological replicate 1 of 3.
|
| Data processing |
Exported fluorescence intensities were normalized in MIDAS (29) using LOWESS (Locally Weighted Scatterplot Smoothing), followed by flip-dye consistency check and in-slide-replicate analysis. The expression profiles were analyzed using EXPANDER, a general microarray analysis software. Please see 'readme.txt' file on the Series record for raw data file - label associations.
|
| |
|
| Submission date |
Oct 26, 2011 |
| Last update date |
Nov 29, 2011 |
| Contact name |
Nir Osherov |
| E-mail(s) |
nosherov@post.tau.ac.il
|
| Phone |
972 3 640 9599
|
| Fax |
972 3 640 9160
|
| Organization name |
Tel-Aviv University
|
| Department |
Medical Microbiology and Immunology
|
| Lab |
Aspergillus Lab Osherov
|
| Street address |
Ramat Aviv Campus
|
| City |
Tel-Aviv |
| ZIP/Postal code |
69978 |
| Country |
Israel |
| |
|
| Platform ID |
GPL14792 |
| Series (1) |
| GSE33254 |
Transcriptional and Proteomic Analysis of the Aspergillus fumigatus ΔprtT Protease-Deficient Mutant |
|
