|
| Status |
Public on Aug 27, 2009 |
| Title |
7 hr 50 uM NE suspension cells |
| Sample type |
RNA |
| |
|
| Source name |
suspension cells of Pseudomonas aeruginosa PA14 WT treated with 50 uM norepinephrine
|
| Organism |
Pseudomonas aeruginosa UCBPP-PA14 |
| Characteristics |
strain: PA14
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A single colony from a fresh plate (<24 hr) plate was inoculated in 25 mL of LB medium and grown overnight at 37 degC at 250 rpm. Two flasks containing 100 mL serum-RPMI medium was inoculated with overnight culture of PA14 WT with a starting OD600 of 0.05. NE (50 uM) was added in one of the flask with equal volume of water in the other. The culture was grown for 7 hours and cell pellets were made subsequently. After breaking the cells with a bead beater, the total RNA was isolated with Qiagen RNeasy mini Kit (Cat# 74104).
|
| Label |
biotin
|
| Label protocol |
Following affymetrix protocol.
cDNA was synthesized first using Promega M-MLV Reverse transcriptase (cat# M1705). After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181).
|
| |
|
| Hybridization protocol |
Prepared hybridization cocktail for Single Probe Array (100 midi Format) with total 130 ul volume including 1X hybrization buffer, 3 nM B2 Control Oligo, 10 mg/mL Herring Sperm DNA, 50 mg/mL BSA, 100 % DMSO and at least 1 ug fragmented and labelled cDNA.
After loading of hybridization cocktail in Affymetrix P.aeruginosa Genome Array, the hybridization was performed at 50ºC, with 60 rpm for 16 hours.
After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS).
|
| Scan protocol |
Following affymetrix protocol.
After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS).
|
| Description |
RNA extracted from suspension cells of Pseudomonas aeruginosa PA14 WT treated with 50 uM norepinephrine for 7 hour in serum-RPMI medium at 37ºC
|
| Data processing |
MAS 5.0 Expression Analysis Default Setting
|
| |
|
| Submission date |
Feb 03, 2009 |
| Last update date |
Aug 27, 2009 |
| Contact name |
Manjunath Hegde |
| E-mail(s) |
coolheggie@gmail.com
|
| Phone |
9798451555
|
| Organization name |
Texas A&M University
|
| Department |
Chemical Engineering
|
| Lab |
Dr. Thomas Wood and Dr. Arul Jayaraman
|
| Street address |
524 Jack E. Brown Building TAMU
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-3122 |
| Country |
USA |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE13326 |
Microarray for suspension cells of PA14 WT with and without NE (50 uM and 500 uM) for 7 h |
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