S. aureus colonies from brain heart infusion (BHI) agar plates were used to inoculate a 3-mL culture in BHI medium and were incubated overnight. The next day, cultures were used to inoculate 200 ml fresh BHI (pH 7.4) to a starting OD600 of 0.05 and incubated with aeration (250 RPM) at 37C. After the bacteria reached an OD600 between 0.3 and 0.4, they were incubated for an additional 30 min (pH maintained at 7.4). At that point, rifampin (200 micrograms per ml) was added to the culture. Twenty one ml aliquots of cells were removed at 0, 2.5, 5, 15 and 30 min post rifampin treatment. One milliliters of cell suspension was serial diluted and plated on both BHI medium and BHI medium containing rifampin. Plates were incubated for 16 hr at 37C and colony forming units per ml were enumerated to measure rifampin resistance. If any rifampin resistant colonies were detected (10-2 dilution) the experiment was discarded and subsequently repeated. Total bacterial RNA was extracted from the remaining 20 ml of each aliquot.
Extracted molecule
total RNA
Extraction protocol
Twenty ml of cells were added to 20 ml ice cold acetone:ethanol (1:1) and stored at -80oC overnight. For RNA isolation, -80oC suspensions were thawed on ice and centrifuged at 5,000 X g at 4oC for 10 min. The supernatant was removed, the cell pellet was resuspended in 1 ml ice cold TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and transferred to a pre-chilled lysing matrix B tube (Q-BIOgene, Irvine, CA). Cells were lysed by shaking in an FP120 shaker (Q-BIOgene) two times at a setting of 4.5 for 20 s. Suspensions were then centrifuged at 13,000 X g at 4oC for 10 min and the supernatant was used for RNA isolation with an RNeasy Mini column, following the manufacturer recommendations for on-the-column DNase treatment and RNA purification (Qiagen, Valencia, CA).
Label
biotin
Label protocol
RNA was reverse transcribed, cDNA fragmented, 3’ biotinylated, mixed with exogenous labeled “spike-in” transcripts and hybridized to S. aureus GeneChips following the manufacturer’s recommendations for antisense prokaryotic arrays (Affymetrix, Santa Clara, CA).
Hybridization protocol
1.5 micrograms (ug) of labeled total RNA was applied to an S. aureus Affymetrix GeneChip hybridized @ 45 degrees Celsius (C) for 16 hr in an Affymetrix hybridization oven. The sample was then washed 60 times in non-stringent buffer (Wash A; 6X SSPE, 0.01% Tween-20) at 45C followed by 60 washes in stringent buffer (Wash B; 1X MES, 25 mM NaCl, 0.01% Tween-20) at 45 C in an Affymetrix Fluidics station (450). The sample was then subjected to the following stains/wash steps: 1. 10 min of Stain 1 (1X MES, 1.2 mg BSA, 6 ug Streptavidin) at 25C, 2. 40 washes with Wash A, 3. 10 min of Stain 2 (1X MES, 1.2 mg BSA, 60 ug Goat-IgG, and 3 ug biotinylated antistreptavidin), 4. 10 min of Stain 3 (1X MES, 1.2 mg BSA, 6 ug Streptavidin-Phycoerythrin), 5. 60 washes with Wash A at 25C.
Scan protocol
The GeneChip was scanned in an Affymetrix 7G scanner.
Description
None
Data processing
The raw signal intensity value and present/absent determinations were calculated based on Affymetrix algorithms (default settings) using GeneChip Operating Software version 1.3
