|
| Status |
Public on Sep 10, 2016 |
| Title |
WP3-cold shock 90 min-rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WP3 cells at mid-exponential phase
|
| Organism |
Shewanella piezotolerans WP3 |
| Characteristics |
culture protocol: WP3 cells were cultured at 20°C in 2216E medium
|
| Growth protocol |
Shewanella strains were cultured in modified marine medium 2216E (5 g/l tryptone, 1 g/l yeast extract, 0.1 g/l FePO4, 34 g/l NaCl) at 20°C, and then were incubated at 0°C for 90 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
The retro-transcription of the 20 μg total RNA was accorded with the Invitrogen manufacturer’s instruction. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction.
|
| |
|
| Channel 2 |
| Source name |
WP3 cells at mid-exponential phase and were incubated at 0°C for 90 min
|
| Organism |
Shewanella piezotolerans WP3 |
| Characteristics |
culture protocol: WP3 cells were cultured at 20℃ in 2216E medium and were incubated at 0℃ for 90 min
|
| Growth protocol |
Shewanella strains were cultured in modified marine medium 2216E (5 g/l tryptone, 1 g/l yeast extract, 0.1 g/l FePO4, 34 g/l NaCl) at 20°C, and then were incubated at 0°C for 90 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
The retro-transcription of the 20 μg total RNA was accorded with the Invitrogen manufacturer’s instruction. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction.
|
| |
|
| |
| Hybridization protocol |
Klenow enzyme labeling strategy was adopted after reverse transcription. Briefly, 20 μg amplified RNA was mixed with 4 μl random hexamers, denatured at 70°C for 5 min, and cooled on ice. Then, 4 μl of first-strand buffer, 2 μl of 0.1M DTT, 1 μl 10mM dNTP, and 1.5 μl SuperScipt Ⅱ (Invitrogen) were added. The mixtures were incubated for 10 min at 25°C, then for 90 min at 42°C. The cDNA products were purified using the PCR purification kit and vacuum evaporated to 10 μl, and then it was mixed with 4 μg random nanomer, heated to 95°C for 3 min, and snap cooled on ice. After that, 10 μl buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (Amersham Biosciences) were added to final concentrations of 120 mM dATP, dGTP, dTTP and 60 mM dCTP, and 40mM Cy-dye. Klenow enzyme (Takara, Japan) was then added, and the reaction was performed for 90 min at 37°C. Labeled cDNA was purified with the PCR purification kit and resuspended in elution buffer.
|
| Scan protocol |
All microarrays were scanned with a LuxScan 10K scanner using microarray scanner 2.3 software (CapitalBio, Beijing, China). We quantified signal intensites of individual spot from the 24-bit TIFF images using SpotData Pro 2.2 (CapitalBio, Beijing, China).
|
| Description |
raw data file: Cold90min-cy3+Cold0min-Cy5.gpr
WP3 cDNA-untreated was labeled with Cy5 and WP3 cDNA-cold shock 90 min was labeled with Cy3
|
| Data processing |
For every microarray slide, there are 16416 spots, which were divided into 48 blocks. For every block, there are 18 columns and 19 rows. 16416=48*18*19. There are 3 spots for a single gene on a single microarray slide, thus we get 3 ratios of the expression level of this gene in 2 samples (rep1, rep2 and rep3). As dye-swaps were performed (label cDNA with different fluorecent dye), we can get another 3 ratios of the expression level of this gene in 2 samples (rep4, rep5 and rep6).
The replicate spots on the array were printed in the same row in adjacent columns. Replicate number was assigned to the spots in ascending column order. For example, WP00027 is printed in block 1, row 2, columns 13, 14 and 15. Replicate 1/4 is column 13, 2/5 is column 14 and replicate 3/6 is column 15.
S-plus software was used for the normalization of microarray data. A spatial and intensity-dependent normalization based on a LOWESS program were used. Normalized data was log transformed and loaded into MAANOVA under R environment for multiple testing, by fitting a mixed effects ANOVA model. Microarray spots with P values <0.01 in the T-test and log2 ratio>1 or <-1 were regarded as differentially expressed genes.
|
| |
|
| Submission date |
Jun 03, 2016 |
| Last update date |
Sep 10, 2016 |
| Contact name |
Huahua Jian |
| E-mail(s) |
jiandy@sjtu.edu.cn
|
| Organization name |
Shanghai Jiao Tong University
|
| Street address |
No.800 Dongchuan Road
|
| City |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
| |
|
| Platform ID |
GPL16568 |
| Series (2) |
| GSE82258 |
Shewanella piezotolerans WP3 wild-type strain cold shock for 90 min |
| GSE82259 |
Shewanella piezotolerans WP3 wild-type strain under cold shock |
|
