|
| Status |
Public on May 03, 2014 |
| Title |
∆cpxR (pBAD33-cpxR) vs ∆cpxR (pBAD33) -1h inducing conditions - Exp 1 - Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
∆cpxR (pBAD33) -1h inducing conditions
|
| Organism |
Vibrio cholerae O1 biovar El Tor str. N16961 |
| Characteristics |
strain: Vibrio cholerae N16961∆cpxR (pBAD33)
|
| Treatment protocol |
Cells were washed, resuspended in LB+arabinose to induce transcription from the PBAD promoter and harvested after 1h of growth at 37°C.
|
| Growth protocol |
V. cholerae N16961 ∆cpxR harboring (pBAD33-cpxR) or (pBAD33) were grown at 37°C in LB+glucose until mid-logarithmic phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
RNA (10 µg) was reverse transcribed in the presence of 0.5 mM dATP, dCTP and dGTP, 0.2 mM dTTP and 0.3 mM aminoallyl dUTP. RNA was then hydrolysed with NaOH, and cDNAs were purified on Miniprep Spin Prep columns (Qiagen). cDNAs were coupled to monoreactive Cy3 and Cy5 (Amersham), then repurified, dried down and resuspended in hybridization buffer (25% formamide, 5X SSC, 0.1% SDS, 0.1 mg ml-1 salmon sperm DNA, 0.2 mg ml-1 yeast tRNA). Probes were denatured at 95∞C before hybridization at 42∞C.
|
| |
|
| Channel 2 |
| Source name |
∆cpxR (pBAD33-cpxR) -1h inducing conditions
|
| Organism |
Vibrio cholerae O1 biovar El Tor str. N16961 |
| Characteristics |
strain: Vibrio cholerae N16961∆cpxR (pBAD33-cpxR)
|
| Treatment protocol |
Cells were washed, resuspended in LB+arabinose to induce transcription from the PBAD promoter and harvested after 1h of growth at 37°C.
|
| Growth protocol |
V. cholerae N16961 ∆cpxR harboring (pBAD33-cpxR) or (pBAD33) were grown at 37°C in LB+glucose until mid-logarithmic phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
RNA (10 µg) was reverse transcribed in the presence of 0.5 mM dATP, dCTP and dGTP, 0.2 mM dTTP and 0.3 mM aminoallyl dUTP. RNA was then hydrolysed with NaOH, and cDNAs were purified on Miniprep Spin Prep columns (Qiagen). cDNAs were coupled to monoreactive Cy3 and Cy5 (Amersham), then repurified, dried down and resuspended in hybridization buffer (25% formamide, 5X SSC, 0.1% SDS, 0.1 mg ml-1 salmon sperm DNA, 0.2 mg ml-1 yeast tRNA). Probes were denatured at 95∞C before hybridization at 42∞C.
|
| |
|
| |
| Hybridization protocol |
Slides were prehybridized at 42°C in 5X SSC, 0.1% SDS, 1% BSA, then briefly rinsed with water and isopropanol. Each array was then hybridized simultaneously to two differentially labelled cDNA probes corresponding to RNA from each strain. After hybridization overnight at 42°C, slides were washed sequentially with (i) 2X SSC, 0.1% SDS; (ii) 0.1X SSC, 0.1% SDS; and (iii) 0.1X SSC.
|
| Scan protocol |
Slides were scanned using a Packard Scanarray 4000 with the PMT adjusted so that fluorescence from Cy3- and Cy5-labelled probes was balanced. Scans were analysed with QUANTARRAY (Packard) to calculate spot intensity and background.
|
| Data processing |
The Quantarray output was normalize by substracting the background intensity for each spot before the results were subjected to global means normalization.
|
| |
|
| Submission date |
Jan 14, 2014 |
| Last update date |
May 04, 2014 |
| Contact name |
Matthew K Waldor |
| Organization name |
HHMI/Brigham and Women’s Hospital
|
| Department |
Division of Infectious Diseases
|
| Lab |
Waldor Lab
|
| Street address |
181 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL3651 |
| Series (1) |
| GSE54081 |
Vibrio cholerae N16961 overexpression of CpxR |
|
