|
| Status |
Public on Feb 20, 2009 |
| Title |
S. aureus_mvaK-IPTG-3 |
| Sample type |
RNA |
| |
|
| Source name |
S. aureus mvaK regulated mutant grown without IPTG in NZYM broth harvested in exponential phase
|
| Organism |
Staphylococcus aureus subsp. aureus RN4220 |
| Characteristics |
RN4220 derivative requiring IPTG for growth
|
| Treatment protocol |
Cells were resuspended in 4 mL water and 8 mL of RNAProtect Bacteria Reagent (QIAGEN, Valencia, CA), reacted for at least 30 minutes, pelleted by centrifugation, then frozen at -80 degrees C until further use.
|
| Growth protocol |
S. aureus were grown overnight in NZYM Broth with 5 ug/mL erythromycin to maintain regulated mutants. Cells were then diluted 5000 fold in fresh NZYM without antibiotic and cultures were grown for 6-8 hours with vigorous shaking at 37 degrees C. In mid-exponential phase an equivalent of 15 mL of culture at OD600 = 1.0 were harvested by centrifugation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cell pellets using the Purescript RNA Isolation Kit (Gentra Systems, Minneapolis, MN) with an initial 0.3 µg/µL lysostaphin (Sigma, St. Louis, MO) treatment to achieve cell lysis. 100 µg of total RNA was treated with 7 units of RNase-free DNase I (QIAGEN, Valencia, CA) and then further purified using the RNeasy Plus Mini Kit (QIAGEN, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA was prepared according to the standard Affymetrix protocol from 15 ug total RNA
|
| |
|
| Hybridization protocol |
Following fragmentation, 3 ug of cDNA were hybridized for 16 hr at 45C on GeneChip S. aureus Genome Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Workstation 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 7G Autoloader
|
| Description |
Gene expression data from exponentially growing cells
mvaK regulated mutant RN4220, - IPTG, rep3
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. Additional normalization was carried out in GeneSpring.
|
| |
|
| Submission date |
Oct 31, 2008 |
| Last update date |
Feb 20, 2009 |
| Contact name |
Carl J. Balibar |
| Organization name |
Novartis Institutes for BioMedical Research
|
| Department |
Infectious Diseases
|
| Lab |
Jianshi Tao
|
| Street address |
500 Technology Sq.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL1339 |
| Series (1) |
| GSE13424 |
Profiling downregulation of the mevalonate pathway in Staphylococcus aureus |
|
