|
| Status |
Public on Apr 11, 2014 |
| Title |
Bsubtilis_KAcetate_t20min_combined_2_replicates |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Bsubtilis_t20min_control
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: 168; PB2
time: 20 minutes of exponential growth at an OD600 of 0.2 in defined minimal medium; sample is part of time series
|
| Treatment protocol |
A sample of 20 ml was withdrawn from the culture at 20 min after t_00min. The cells were collected using a vacuum-filtering set-up, immediately quenched in liquid nitrogen and stored at -80 ˚C prior to RNA extraction. The whole procedure took no longer than 50 s.
|
| Growth protocol |
An exponentially growing culture of B. subtilis WT strain 168 was inoculated in a well-controlled batch-fermentor (500 ml working volume) to an OD600 of 0.05. The culture was grown at 37 °C in defined medium at pH 6.4 with an aeration rate of 0.5 liters/min and vigorous stirring (200 rpm). At an OD600 of 0.2 (t_00min) the first sample of the time-series was taken.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Fast RNA pro Blue kit (Qbiogene) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Direct incorporation: Random priming of SSII RT to incorporate Cy5- or Cy3-labeled dUTP, followed by NaOH hydrolysis of RNA
|
| |
|
| Channel 2 |
| Source name |
Bsubtilis_t20min_KAcetate_treatment
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: 168; PB2
time: 20 minutes of growth in defined minimal medium after addition of 25 mM potassium acetate at an OD600 of 0.2; sample is part of time series
|
| Treatment protocol |
A sample of 20 ml was withdrawn from the culture at 20 min after addition of 25 mM potassium acetate. The cells were collected using a vacuum-filtering set-up, immediately quenched in liquid nitrogen and stored at -80 ˚C prior to RNA extraction. The whole procedure took no longer than 50 s.
|
| Growth protocol |
An exponentially growing culture of B. subtilis WT strain 168 was inoculated in a well-controlled batch-fermentor (500 ml working volume) to an OD600 of 0.05. The culture was grown at 37 °C in defined medium at pH 6.4 with an aeration rate of 0.5 liters/min and vigorous stirring (200 rpm). At an OD600 of 0.2 (t_00min), the culture was stressed with 25 mM potassium acetate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Fast RNA pro Blue kit (Qbiogene) following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Direct incorporation: Random priming of SSII RT to incorporate Cy5- or Cy3-labeled dUTP, followed by NaOH hydrolysis of RNA
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed in an automated slide processor (GE Healthcare) for 16 h at 37°C
|
| Scan protocol |
Microarrays were scanned in an Agilent DNA MicroArray Scanner (Agilent Technologies, Palo Alto, CA,USA) at 100 % PMT and 100% laser power
|
| Description |
20 min treatment (potassium acetate) vs. control (average of 2 biological replicates)
|
| Data processing |
Data supplied is log2 ratio of normalized and filtered 2 biological replicates, each containing 2 technical replicates (duplicate spots on the microarray and duplicate hybridization). Raw data and required information is supplied as supplementary file. The data was normalized in J-Express Pro 2.7 software (MolMine AS) using a global LOWESS (locally weighted scatterspot smoothing) algorithm. To avoid extreme intensity ratios, low intensity fluorescence data was floored at a value corresponding to a signal-to-noise ratio of 2.0. The data was averaged, log2 transformed and missing values were replaced by the average of the closest values. Genes with more than one missing value in the time-series were omitted. Since the variation in differential expression measurements depends on the fluorescent signal intensity (smaller variation at higher- and larger variation at lower fluorescence intensity levels), we applied an intensity-dependent method to identify differentially expressed genes. A sliding window of 50 genes was selected to calculate a Z-score from the local mean and standard deviation using the data in the R-I plot (log10(Cy5*Cy3) vs. log10(Cy5/Cy3). Genes more than 1.96 standard deviations away from the local average (|Z| > 1.96) were considered differentially expressed. This corresponds to a confidence level of 95 %. Genes that showed significant expression at t = 0 min were excluded from further analysis, unless the gene showed opposite significant expression in at least one of the other time-points. After the processing of the microarray data 3909 genes remained for each time-point, of which 459 were found to be significantly expressed. After the processing of the microarray data 3746 and 3685 genes of the total number of 4100 genes present on the array remained for each time-point, of which 503 and 582 were found to be significantly expressed in the potassium acetate and CCCP treatments, respectively.
|
| |
|
| Submission date |
Feb 14, 2014 |
| Last update date |
Apr 11, 2014 |
| Contact name |
Alexander Ter Beek |
| E-mail(s) |
alexander.terbeek@gmail.com
|
| Organization name |
University of Amsterdam - Swammerdam Institute for Life Sciences
|
| Department |
Molecular Biology & Microbial Food Safety
|
| Lab |
prof. Stanley Brul
|
| Street address |
Science Park 904
|
| City |
Amsterdam |
| ZIP/Postal code |
1098XH |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6257 |
| Series (1) |
| GSE55051 |
Time-series of transcriptome analysis of Bacillus subtilis response to 25 mM potassium acetate and 0.85 µM CCCP |
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