|
| Status |
Public on Jan 01, 2016 |
| Title |
Δpgm vs. Δpgm ΔypeIR rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Δpgm
|
| Organism |
Yersinia pestis |
| Characteristics |
genotype/variation: Δpgm
strain: CO92
|
| Treatment protocol |
Microarray studies were performed by comparing strains R88 and R109 grown at a mid logarithmic phase.
|
| Growth protocol |
An overnight culture was diluted 1:100 and grown at 30°C in HIB 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 1 ml of Trizol (Ambion) and were then frozen. RNA was extracted from frozen cell pellets using the Trizol reagent protocol. The RNA was treated with DNase I (Ambion) at 37°C for 30 min to remove contaminating genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30.
|
| Label |
Alexa555
|
| Label protocol |
Target generation and labeling were performed as described (Carruthers MD and Minion C. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102).
|
| |
|
| Channel 2 |
| Source name |
Δpgm ΔypeIR
|
| Organism |
Yersinia pestis |
| Characteristics |
genotype/variation: Δpgm ΔypeIR
strain: CO92
|
| Treatment protocol |
Microarray studies were performed by comparing strains R88 and R109 grown at a mid logarithmic phase.
|
| Growth protocol |
An overnight culture was diluted 1:100 and grown at 30°C in HIB 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 1 ml of Trizol (Ambion) and were then frozen. RNA was extracted from frozen cell pellets using the Trizol reagent protocol. The RNA was treated with DNase I (Ambion) at 37°C for 30 min to remove contaminating genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30.
|
| Label |
Alexa647
|
| Label protocol |
Target generation and labeling were performed as described (Carruthers MD and Minion C. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102).
|
| |
|
| |
| Hybridization protocol |
Corresponding equal amounts (1.5 µg) of Alexa 555- or Alexa 647-labelled cDNA targets were mixed and dried using a Thermo Scientific Savant DNA SpeedVac Concentrator. Targets were suspended in 60 µl hybridization buffer (40% formamide, 5x SSC, 0.1% SDS, 0.6 µg/µl Salmon Sperm DNA), incubated at 95°C for 5 min, centrifuged (10,000 x g, 4 min), and kept at room temperature until placed in a hybridization chamber. Hybridization lasted for 16 h at 42°C and washed in a series of wash buffers (2x saline-sodium citrate (SSC) - 0.1% SDS; 0.1x SSC – 0.1% SDS, and 0.1x SSC) and dried by centrifugation at 1500 x g for 30 s.
|
| 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 |
The strain Δpgm is the Y.pestis CO92 pigmentation-negative mutant R88. The Δpgm ΔypeIR ΔyspIR is R114 strain.
|
| Data processing |
Data acquisition, normalization and data analysis have been described (Carruthers MD and Minion C. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102).
|
| |
|
| Submission date |
Jun 30, 2011 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Chris Minion |
| E-mail(s) |
fcminion@iastate.edu
|
| Phone |
515-294-6347
|
| Fax |
515-294-8500
|
| Organization name |
Iowa State University
|
| Department |
VMPM
|
| Street address |
1130 Vet Med
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL9009 |
| Series (2) |
| GSE30332 |
Global gene expression analysis of Yersinia pestis Ype AHL quorum sensing at 30°C |
| GSE30373 |
Global gene expression analysis of *Yersinia pestis* AHL quorum sensing |
|
