|
| Status |
Public on Nov 26, 2008 |
| Title |
NTHi 4h; 1740174013_F |
| Sample type |
RNA |
| |
|
| Source name |
Lung homogenate, 4h NTHi treatment
|
| Organism |
Mus musculus |
| Characteristics |
C57BL/6
|
| Treatment protocol |
The NTHi cell pellet was washed and resuspended in 20 ml 0.9% sodium chloride solution. This suspension was passed three times through an EmulsiFlex C5 cell disruptor (Avestin) at greater then 10,000 psi, then diluted to 4-5 mg/ml in 0.9% sodium chloride solution by bicinchoninic assay (Pierce) and centrifuged at 15,000 x g for 10 min. The supernatant was collected, the protein concentration was adjusted to 2.5 mg/ml, and the lysate was sterilized by passage through a 0.2 µm filter and frozen in 8 ml aliquots at -80ºC. For treatment, a thawed aliquot was placed in an AeroMist CA-209 nebulizer driven by 10 l/min 5% CO2 in air for 20 min, resulting in aerosolization of 4 ml of lysate, with the protein concentration in residual lysate
|
| Growth protocol |
Frozen stock (1 x 107 CFU/ml) of non-typeable Haemophilus influenzae (NTHi) was grown on chocolate agar at 300 ul per 10 cm plate (Remel) for 24 h at 37ºC in 5% CO2, then harvested and incubated for 16 h in 1 l brain-heart infusion broth (Acumedia) supplemented with 3.5 ug/ml NAD (Sigma-Adrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA extracted using Qiagen Rneasy per manufacturers instructions. cRNA amplification using Ambion total prep kit per manufacturers instructions.
|
| Label |
Cy3
|
| Label protocol |
labeling with Cy3-Streptavidin per Illumina instructions.
|
| |
|
| Hybridization protocol |
cRNA was hybridized to BeadChips per Illumina instructions.
|
| Scan protocol |
Chips were scanned using level 3 signal using a BeadStation 500 scanner.
|
| Description |
Gene expression in C57BL/6 mouse lungs after exposure to aerosolized NTHi lysate
|
| Data processing |
Data was processed using Illumina BeadStudio v3. Data generated both with rank invariant normalization, using sham group as the reference group.
|
| |
|
| Submission date |
Nov 25, 2008 |
| Last update date |
Nov 25, 2008 |
| Contact name |
Scott E Evans |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Texas-M.D. Anderson Cancer Center
|
| Department |
Pulmonary Medicine - Unit 1462
|
| Street address |
1400 Holcombe Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL6105 |
| Series (1) |
| GSE13740 |
Gene expression in C57BL/6 mouse lungs after exposure to aerosolized NTHi lysate |
|
