|
| Status |
Public on Nov 15, 2011 |
| Title |
Mutant time2 rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mutant
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
strain: 4074
genotype variation: luxS negative
|
| Treatment protocol |
cultured in TSB medium to middle exponential phase
|
| Growth protocol |
A. pleuropneumoniae strains were cultured in TSB medium supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 10% (v/v) filtered cattle serum at 37°C. For samples from the mutant complemented with DPD, 100μM and 25μM DPD were added into the medium before early exponential phase as well as one hour before late exponential phase respectively. The samples were collected from early, middle, late exponential phase and stationary phase respectively and the total RNA were extracted using Trizol reagent according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA were extracted using RNA-Solv Reagent (Omega) according to the manufacturer’s instructions. Total RNA were purified using QIAGEN RNeasy Mini Kit (QIAGEN). 2μg RNA were reverse-transcribed into cDNA using T7 promotor primer.
|
| Label |
cy3
|
| Label protocol |
cDNA were transcribed into cRNA using T7 RNA Polymerase and aaUTP (Ambion). cRNA were purified using QIAGEN RNeasy Mini kit (QIAGEN). 4μg cRNA were combined with DMSO and 0.3M NaHCO3 and then were added into Cy3 NHS ester (GE healthcare). The mixture were incubated at 25°C for 1 hour. 4M Hydroxylamine were added and incubated at 25°C for 15min. The labeled cRNA were purified using QIAGEN RNeasy Mini kit (QIAGEN).
|
| |
|
| Hybridization protocol |
Hybridization was performed using Gene Expression Hybridization Kit (Agilent). 1μg of cRNA were mixed with blocking agent, fragmentation buffer and incubated at 60°C for 30 min for fragmentation. GEx Hybridization Buffer were added and 100 μl mixture were hybridized at 65°C for 17 hours with 10 rpm rotation. The arrays were washed two times using wash buffer 1 and 2 from Gene Expression Wash Buffer Kit (Agilent).
|
| Scan protocol |
Arrays were scanned using Agilent Microarray Scanner System G2565BA (Agilent) with resolution of 5μm. The scanner uses 100% and 10% PTM respectively and combines the two data automatically.
|
| Description |
Two biological repetitions were combined
|
| Data processing |
We used and submitted only one probe signal (the probe that near the 3 end of the gene) for each gene in this study. The 2477 rows in the sample data table represented 2477 ORFs on the array. The data were analyzed using Feature Extraction Software (Agilent). The signal intensity were log2 normalized.
|
| |
|
| Submission date |
Apr 12, 2010 |
| Last update date |
Nov 15, 2011 |
| Contact name |
Lu Li |
| E-mail(s) |
sakura.tree@163.com, xuzf@mail.hzau.edu.cn, xuzhuofei@sohu.com, kobe2071@tom.com
|
| Organization name |
Huazhong Agricultural University
|
| Street address |
Shizishan Street 1
|
| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL9691 |
| Series (1) |
| GSE21300 |
Expression data of Actinobacillus pleuropneumoniae 4074, the ΔluxS mutant and AI-2 supplemented ΔluxS mutant |
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