S. aureus strains were cultured at 37C to mid exponential phase OD600=1 (~2 hrs) in tryptic soy broth.
Extracted molecule
total RNA
Extraction protocol
Microarray analysis of S. aureus gene expression. Aliquots of cells harvested during exponential phase (3 x 108 CFU/ml) and stationary phase (4 x 109 CFU/ml) were pelleted by centrifugation at 8000 x g for 5 minutes at 4ºC. Each pellet was washed in an equal volume of TE buffer (10mM TrisHCl, 1mM EDTA [pH=8]), resuspended to 109 CFU/ml in TE buffer containing 100μg lysostaphin (Sigma-Aldrich, St. Louis, MO) and 250 μg lysozyme (Sigma-Aldrich, St. Louis, MO) and incubated at 37ºC for 15 minutes. Total bacterial RNA was isolated using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen Sciences, Maryland). Contaminating DNA was removed incubating the total bacterial RNA with RNase-free DNase I (Ambion Inc., Austin, USA) 30U/100μg of total RNA, for 1h at 37°C. RNA was then re-purified using RNeasy Mini columns (Qiagen Sciences). The amount of RNA recovered was determined using a NanoDrop Fluorospectometer (NanoDrop Technologies, Wilmington,DE) and the purity and quality of RNA were determined using a Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). The microarray experiment was performed by NimbleGen Systems Inc., Madison, WI. RNA was converted to cDNA, labeled and hybridized to a 24 mer Staphylococcus aureus NCTC 8325 gene expression array (2641 genes; 190,000 probes).
Label
Cy3
Label protocol
Samples were labeled by Nimblegen Systems, Inc. www.nimblegen.com
Hybridization protocol
Hibridization was performed by Nimblegen Systems, Inc. www.nimblegen.com
Scan protocol
Scan was performed by Nimblegen Systems, Inc.
Description
exponential phase
Data processing
The data was normalized using quantile normalization and gene calls generated using RMA (Robust Multichip Average), tools available through Bioconductor project (http://www.bioconductor.org).
