|
| Status |
Public on Mar 11, 2015 |
| Title |
Over-expression Rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FKH2-HA
|
| Organism |
Candida albicans |
| Characteristics |
morph: yeast
genotype: BWP17 FKH2/FKH2-HA:URA3
|
| Treatment protocol |
For hyphal induction, cells were washed in sterile water and diluted to OD600 = 0.6 in pH 7.0 media supplemented with 10-20% fetal calf serum (FCS) and incubated at 37°C.
|
| Growth protocol |
C. albicans cells were routinely grown in 1% yeast extract, 2% peptone and either 2% glucose (YEPD) or galactose (YEPG), or in 0.67% yeast nitrogen base without amino acids and 2% glucose (GMM) with appropriate amino acids for auxotrophic mutants.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy minikit (QIAGEN) following the manufacturer’s instructions for yeast mechanical disruption, using a TOMY Microsmash (TOMY Digital Biology, Tokyo JP). Total RNA was treated with DNase I (Roche) before RNA-cleanup using the Rneasy kit
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA was reverse-transcribed with either Cy3 (control) or Cy5 (experimental) dyes
|
| |
|
| Channel 2 |
| Source name |
PGAL1-FKH2-GFP
|
| Organism |
Candida albicans |
| Characteristics |
morph: yeast
genotype: BWP17 FKH2/FKH2/PGAL1-FKH2-GFP:URA3
|
| Treatment protocol |
For hyphal induction, cells were washed in sterile water and diluted to OD600 = 0.6 in pH 7.0 media supplemented with 10-20% fetal calf serum (FCS) and incubated at 37°C.
|
| Growth protocol |
C. albicans cells were routinely grown in 1% yeast extract, 2% peptone and either 2% glucose (YEPD) or galactose (YEPG), or in 0.67% yeast nitrogen base without amino acids and 2% glucose (GMM) with appropriate amino acids for auxotrophic mutants.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy minikit (QIAGEN) following the manufacturer’s instructions for yeast mechanical disruption, using a TOMY Microsmash (TOMY Digital Biology, Tokyo JP). Total RNA was treated with DNase I (Roche) before RNA-cleanup using the Rneasy kit
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA was reverse-transcribed with either Cy3 (control) or Cy5 (experimental) dyes
|
| |
|
| |
| Hybridization protocol |
Not provided
|
| Scan protocol |
Arrays were scanned with an Axon GenePix 4000B scanner
|
| Data processing |
Microarray data was analysed using the limma package. Arrays were background normalised using the “normexp” method and spots with an intensity of less than 50 over background in both channels were removed. Arrays were then normalized using the “print-tip loess” method. Within array replicate spots were averaged. Differentially expressed genes between wild type, fkh2(6A) and fkh2ΔΔ yeast samples were assessed using empirical Bayes moderated t-tests. For hyphal samples we fitted a linear model with indicator variables for genotype and used moderated t-statistics to test the significance of the coefficients for kinase mutant and deletion genotype, as well as the significance of the difference between these coefficients. False discovery rate was assessed across all tests simultaneously using the method of Benjamini and Hochberg [71]. Where multiple probes targeted the same gene, the least significant was selected.
The results presented in the Matrix table are the last stage at which the results can easily be presented in matrix table format. The whole oligo set is printed twice on the chip, and these duplicates have been averaged. Some genes are represented by multiple spots (either as independent or repeated oligos), handling of these replicates is handled on a test-by-test basis in the p-value summarisation of the data, and is different for different models tested.
|
| |
|
| Submission date |
Dec 19, 2014 |
| Last update date |
Mar 12, 2015 |
| Contact name |
Ian Sudbery |
| E-mail(s) |
i.sudbery@sheffield.ac.uk
|
| Organization name |
University of Sheffield
|
| Department |
Molecular Biology and Biotechnology
|
| Lab |
Sudbery Lab for Computational Genomics
|
| Street address |
Firth Court, Western Bank
|
| City |
Sheffield |
| ZIP/Postal code |
S11 8HG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19574 |
| Series (1) |
| GSE64383 |
Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis. |
|
