|
| Status |
Public on Nov 14, 2012 |
| Title |
wt-htp85-b |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wt-htp85-b
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4742
genotype: wt
|
| Growth protocol |
Yeast growth for expression profiling in Tecan platereader v1.0
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
yeast HTP RNA isolation for robot v2.0
|
| Label |
Cy5
|
| Label protocol |
robot amplification and labeling v1.0
|
| |
|
| Channel 2 |
| Source name |
ref1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4742
genotype: ref1
|
| Growth protocol |
yeast-deleteome[grow]
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
yeast RNA isolation for robotic amplification v1.0
|
| Label |
Cy3
|
| Label protocol |
robot amplification and labeling v1.0
|
| |
|
| |
| Hybridization protocol |
Tecan HS4800 hybridization
|
| Scan protocol |
scanning of slides using Agilent G256BA
Imagene feature extraction
|
| Description |
Strains were streaked from -80C stocks onto plates and grown for 3-5 days depending on growth rate. Liquid cultures were inoculated with independent colonies and grown overnight in Synthetic Complete (SC) medium: 2gr/l Drop out mix Complete and 6.71gr/l Yeast Nitrogen Base without AA, Carbohydrate and w/AS (YNB) from US Biologicals (Swampscott, USA) with 2percent D-glucose. Overnight cultures were diluted to an OD600 of 0.15 in 1.5 ml fresh medium and grown at 30C in a 24 well plate in a Tecan Infinite F200 under continuous shaking. Growth curves were made for the mutant cultures (two cultures from two isolates) as well as for two wildtype (wt) inoculates, grown in parallel. Mutant and wt cells were harvested by centrifugation (6100 rpm, 3 min) at mid-log phase at an OD600 of 0.6, and pellets were immediately frozen in liquid nitrogen after removal of supernatant. S. cerevisiae BY4742 deletion mutants and the wild-type strains were cultured in SC Medium (Synthetic Complete; 4gr per 2l Drop out mix 13.42gr per 2l YNB(US biologicals) with 2percent D-glucose)under agitation (230rpm), at 30C. The cells were collected at midlog.
|
| Data processing |
Normalization was done using print-tip LOESS as described in (Yang et al. 2002), by estimating the LOESS curve for all gene probes using no background substraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve. Signals were corrected for dye bias using intrinsic gene-specific dye biases estimated from a set of wild-type wild-type hybridizations, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009.
|
| |
|
| Submission date |
Nov 09, 2012 |
| Last update date |
Nov 14, 2012 |
| Contact name |
Patrick Kemmeren |
| Organization name |
UMC Utrecht
|
| Department |
Department of Molecular Cancer Research
|
| Lab |
Holstege Lab
|
| Street address |
Universiteitsweg 100
|
| City |
Utrecht |
| State/province |
Utrecht |
| ZIP/Postal code |
3584 CG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11232 |
| Series (2) |
| GSE42217 |
yeast wt pool - plate reader |
| GSE45115 |
Expression profiling of 376 wildtypes to assess day-to-day variance |
|
