|
| Status |
Public on Sep 30, 2015 |
| Title |
lung_FT LVS_LPS_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
FT LVS LPS
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
agent: FT LVS LPS
time p.i.: 20hrs
|
| Treatment protocol |
, mice were first exposed to either aerosolized PBS, Francisella tularensis subsp tularensis (FT SchuS4), the live vaccine strain of Francisella tularensis subsp. holarctica (FT LVS), or Francisella novicida (FN). Approximately 18hrs post-exposure, infected and control animals were exposed to aerosolized E. coli LPS, a potent TLR4 agonist. [25]. Lung samples were harvested 2.5 hrs later to measure changes in host transcriptome using microarray technology.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from harvested lungs using Trizol/phenol chloroform extraction followed by precipitation. RNA quality assessed using Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.3 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression profiling after 20hrs LVS LPS
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Feb 11, 2015 |
| Last update date |
Sep 30, 2015 |
| Contact name |
kathie walters |
| E-mail(s) |
kwalters@systemsbiology.org
|
| Organization name |
Institute for Systems Biology
|
| Street address |
401 Terry Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE65871 |
Prior infection with Type A Francisella tularensis antagonizes the pulmonary transcriptional response to an aerosolized TLR4 agonist |
|
