Two strains, Pgm- as treatment and 3XQS as control.
Growth protocol
An overnight culture was diluted 1:100 and grown at 30°C in BHI broth until an optical density 600 nm of 1.0 is reached.
Extracted molecule
total RNA
Extraction protocol
Cells were pelleted and immediately resuspended in 0.5 ml of RNA Protect Reagent (Qiagen, Valencia, CA). RNA was extracted from frozen cell pellets using the RNeasy Mini kit (Qiagen). The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 30 min to remove the genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30(Millipore, Billerica, MA).
Label
Cy 3
Label protocol
Target generation and labeling were performed as described (Carruthers, M. D., and C. Minion. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102).
Two strains, Pgm- as treatment and 3XQS as control.
Growth protocol
An overnight culture was diluted 1:100 and grown at 30°C in BHI broth until an optical density 600 nm of 1.0 is reached.
Extracted molecule
total RNA
Extraction protocol
Cells were pelleted and immediately resuspended in 0.5 ml of RNA Protect Reagent (Qiagen, Valencia, CA). RNA was extracted from frozen cell pellets using the RNeasy Mini kit (Qiagen). The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 30 min to remove the genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30(Millipore, Billerica, MA).
Label
Cy 5
Label protocol
Target generation and labeling were performed as described (Carruthers, M. D., and C. Minion. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102).
Hybridization protocol
Microarray hybridization and post washes were performed using a Lucidea Slidepro Hybridization Station (GE Healthcare). Corresponding equal amounts (1.5 µg) of Cy3- or Cy5-labelled cDNA targets were mixed and dried using a Thermo Scientific Savant DNA SpeedVac Concentrator (Waltham, MA). Targets were suspended in 225 μl Long Oligo hybridization solution (Corning, Inc., Corning, NY), incubated at 95°C for 5 min, centrifuged (10,000 x g, 4 min), and kept at room temperature until injected into the hybridization station. Hybridization lasted for 16 hours at 42°C and washed in a series of wash buffers (2x saline-sodium citrate (SSC), 0.1% SDS; 1x SSC, and 0.1x SSC) by the hybridization station and dried by centrifugation at 1500 x g for 30 secs.
Scan protocol
Arrays scanned on a ProScanArray HT scanner (Perkin Elmer, Wellesley, MA) three times with varying photomultiplier tube gain and laser power settings.
Description
Biological replicate 5 of 6. Control: 3XQS. Treatment: Pgm-.
Data processing
Background correction, compression, normalization and fitting each probe with a mixed model were conducted as previously described (Madsen, M. L., D. Nettleton, E. L. Thacker, and F. C. Minion. 2006.Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152:937-944) excluding slide region and slide-by-region interaction effects. intensity = mu + trt + dye + slide
