Cultures were initiated from overnight cultures in MHB into fresh MHB at 1:100 and grown to OD600=0.4.
Extracted molecule
total RNA
Extraction protocol
Samples were added to RNA Protect (Qiagen, Valencia, CA) and processed according to the manufacturer's instructions. Cells were harvested by centrifugation (8,000 X g, 20 min, 4C) and then resuspended in 1 ml Trizol (Invitrogen, Carlsbad, CA) and processed in an FP120 FastPrep cell disruptor (MP Biomedicals, Irvine, CA). Chloroform was subsequently added to the lysates, followed by centrifugation (16,000 X g, 15 min, 4C) and RNA was precipitated 1:1 (vol/vol) in 100% ethyl alcohol. The RNA was then purified using the RNeasy™ kit (Qiagen) according to the manufacturer's instructions. Contaminating DNA was removed from purified RNA using DNAfree (Ambion, Austin, TX). cDNA was produced using SuperScript II Reverse Transcriptase (Invitrogen) from 2 µg of total RNA combined with random hexamers, 0.25 mM deoxynucleoside triphosphate, and 0.25 mM aminoallyl-dUTP.
Label
Cy3,Cy5
Label protocol
Amino-allyl labeled cDNA was dried and then suspended in 0.1 M sodium carbonate (pH 9.3) and coupled with either Cy3 or Cy5 (swapped between balanced samples, N=4). Uncoupled dye was removed by column purification (Qiagen).
Cultures were initiated from overnight cultures in MHB into fresh MHB at 1:100 and grown to OD600=0.4.
Extracted molecule
total RNA
Extraction protocol
Samples were added to RNA Protect (Qiagen, Valencia, CA) and processed according to the manufacturer's instructions. Cells were harvested by centrifugation (8,000 X g, 20 min, 4C) and then resuspended in 1 ml Trizol (Invitrogen, Carlsbad, CA) and processed in an FP120 FastPrep cell disruptor (MP Biomedicals, Irvine, CA). Chloroform was subsequently added to the lysates, followed by centrifugation (16,000 X g, 15 min, 4C) and RNA was precipitated 1:1 (vol/vol) in 100% ethyl alcohol. The RNA was then purified using the RNeasy™ kit (Qiagen) according to the manufacturer's instructions. Contaminating DNA was removed from purified RNA using DNAfree (Ambion, Austin, TX). cDNA was produced using SuperScript II Reverse Transcriptase (Invitrogen) from 2 µg of total RNA combined with random hexamers, 0.25 mM deoxynucleoside triphosphate, and 0.25 mM aminoallyl-dUTP.
Label
Cy5,Cy3
Label protocol
Amino-allyl labeled cDNA was dried and then suspended in 0.1 M sodium carbonate (pH 9.3) and coupled with either Cy3 or Cy5 (swapped between balanced samples, N=4). Uncoupled dye was removed by column purification (Qiagen).
Hybridization protocol
Arrays were blocked in 1% SDS, 5 X SSC and 1 mg/ml BSA at 42C for 1 h. Arrays were then washed 2X for 5 min in 0.1X SSC and 2X for 30 sec in sterile double-distilled water. Arrays were hybirdized to Cy-labeled cDNAs denatured at 05C in 10 mM EDTA for 5 min and mixed with 40 ul hybridization buffer (Ambion) before loading under a caverslip on the array and sealing in hybridization cassettes. Hybridization was carried out at 47C for 18 h. Afterwards, array slides were washed in 2X SSC, 0.5% SDS at 37C for 5 min, followed by stringency washes 2X in 0.1X SSC at 37C for 2.5 min each, and 2X in 0.1X SSC at room temperature for 2.5 min each.
Scan protocol
Arrays were scanned using an Axon 4000b, and images analyzed using GenePix 6.0.
Description
Analysis used CB1118 RNA as control samples for comparison to CB1541 S. aureus RNA samples.
Data processing
Intensity of spots are quantified by using Spotfinder. Low intensity filter in MIDAS is used to filter out points with intensity less than 1000. Array intensity data was log2-transformed, and normalized using the Lowess algorithm. Statistical analysis was performed using a significance analysis of microarrays (SAM, MultiExperiment Viewer, ver. 4.0) unpaired contrast. A d-statistic was calculated for each gene based on repeated permutations, and a false discovery rate FDR of 0.01 was used to assign a critical cutoff for significance.
Additional Routes to Staphylococcus aureus Daptomycin Resistance as Revealed by Comparative Genome Sequencing, Transcriptional Profiling, and Phenotypic Studies
