|
| Status |
Public on Jun 04, 2011 |
| Title |
untreated_wt_vs_lpdA_03 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FLS186_lpdA_RNA_03
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: FLS186_lpdA
developmental stage: late log
|
| Treatment protocol |
Experimental cultures were treated with the NO- donor SperNO (1mM) for 15 min.
|
| Growth protocol |
Overnight cultures were diluted 1:100 in fresh BHI medium, incubated at 37°C with constant agitation, and allowed to reach an OD600 of 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from 1 mL culture aliquots of treated and untreated cells was stabilized by the addition of 5 ml RNAProtect (Qiagen). Total RNA was isolated using the Qiagen RNeasy midi kit according to the manufacturer’s guidelines.
|
| Label |
Cy3
|
| Label protocol |
For synthesis of cDNA, total RNA was first annealed with N6 random hexamers at 70°C for 10 min and allowed to cool on ice. The cDNA was synthesized in a reaction containing a nucleotide mix with a 2:3 ration of aminoallyl-dUTP:dTTP (Sigma-Aldrich, St. Louis, MO), Super Script- II reverse transcriptase (Invitrogen) and 50 μg total RNA incubated at 42°C overnight. Residual RNA was hydrolyzed in a final concentration of 0.3M NaOH and 0.125M EDTA at 65°C for 10 min, then neutralized with 1M Tris-HCL pH8.0. Unincorporated nucleotides were removed using a Qiaquick PCR purification kit with modified wash buffer: 5mM KPO4 pH8.0 and 80% ethanol. Aminoallyl-labled cDNA was eluted from the columns in water and concentrated on a SpeedVac. Cy5 and Cy3 dyes were coupled to the aminoallyl-labeled cDNA by resuspending the dried cDNA in 0.1M sodium carbonate (pH9.3) with either Cy5 or Cy3 dyes (GE HealthCare)dissolved in DMSO for 1 hr at room temperature. The reaction was neutralized by theaddition of 3M sodium acetate (pH5.2) and excess dye removed by purification on a Qiaquick PCR column using the manufacturer’s wash buffer.
|
| |
|
| Channel 2 |
| Source name |
14028s_wt_RNA_03
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: 14028s_wt
developmental stage: late log
|
| Treatment protocol |
Experimental cultures were treated with the NO- donor SperNO (1mM) for 15 min.
|
| Growth protocol |
Overnight cultures were diluted 1:100 in fresh BHI medium, incubated at 37°C with constant agitation, and allowed to reach an OD600 of 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from 1 mL culture aliquots of treated and untreated cells was stabilized by the addition of 5 ml RNAProtect (Qiagen). Total RNA was isolated using the Qiagen RNeasy midi kit according to the manufacturer’s guidelines.
|
| Label |
Cy5
|
| Label protocol |
For synthesis of cDNA, total RNA was first annealed with N6 random hexamers at 70°C for 10 min and allowed to cool on ice. The cDNA was synthesized in a reaction containing a nucleotide mix with a 2:3 ration of aminoallyl-dUTP:dTTP (Sigma-Aldrich, St. Louis, MO), Super Script- II reverse transcriptase (Invitrogen) and 50 μg total RNA incubated at 42°C overnight. Residual RNA was hydrolyzed in a final concentration of 0.3M NaOH and 0.125M EDTA at 65°C for 10 min, then neutralized with 1M Tris-HCL pH8.0. Unincorporated nucleotides were removed using a Qiaquick PCR purification kit with modified wash buffer: 5mM KPO4 pH8.0 and 80% ethanol. Aminoallyl-labled cDNA was eluted from the columns in water and concentrated on a SpeedVac. Cy5 and Cy3 dyes were coupled to the aminoallyl-labeled cDNA by resuspending the dried cDNA in 0.1M sodium carbonate (pH9.3) with either Cy5 or Cy3 dyes (GE HealthCare)dissolved in DMSO for 1 hr at room temperature. The reaction was neutralized by theaddition of 3M sodium acetate (pH5.2) and excess dye removed by purification on a Qiaquick PCR column using the manufacturer’s wash buffer.
|
| |
|
| |
| Hybridization protocol |
overnight hyb on Corning UltraGAPS slides at 42°C in 25% formamide / 5xSSC / 0.1% BSA
|
| Scan protocol |
Perkin Elmer ScanArray 5000 scanner, image acquisition with ScanArray Express 3.0.1 software
|
| Description |
biological replicate 1_3 of 1_3
|
| Data processing |
Spot intensities were quantified using DigitalGENOME (molecularware) spot-finding software. Intensities were then normalized by 75th percentile scaling before building signal ratios.
|
| |
|
| Submission date |
Jun 03, 2011 |
| Last update date |
Jun 04, 2011 |
| Contact name |
Michael McClelland |
| E-mail(s) |
[email protected]
|
| Phone |
858-336-9554
|
| Organization name |
University of California, Irvine
|
| Department |
Microbiology & Molecular Genetics
|
| Street address |
132 Med Surge I
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL11279 |
| Series (1) |
| GSE29735 |
Similar effects of an NO challenge and an lpdA knockout on transcription of Salmonella enterica sv Typhimurium 14028 |
|
