|
| Status |
Public on Oct 31, 2011 |
| Title |
timepoint 36 vs timepoint 36 #2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
timepoint 36
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: strain 168
|
| Growth protocol |
Wild-type Bacillus subtilis 168 (trpC2; Anagnostopoulos and Spizizen, 1961) was grown in TY medium (10 g/L trypton, 5 g/L yeast extract, 5 g/L NaCl) in batch mode at 37°C in a 16 L fermentor using a working volume of 12 L. Cultures were aerated at 40 L/min and 400 rpm. The pH and oxygen levels were monitored, but were allowed to fluctuate during the batch culture incubation period. To prevent foaming, 0.3% antifoam A (SIGMA) was added on an as-needed basis. Cultures were inoculated to an OD600 of approximately 0.01 from synchronized exponentially grown pre-cultures grown at 37°C in TY and frozen in 10% glycerol (Lulko et al., 2007). Growth was monitored by measurement of the OD at 600 nm. As soon as the culture reached an OD600 of 0.1 (typically after around 75 min.), samples were taken every 10 minutes for optical density measurements and RNA isolation. In total 40 timepoints were sampled. Cells were harvested by centrifugation (14.000 rpm, 4°C, 1 min.) and the pellet was immediately frozen in liquid nitrogen and stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with the High Pure RNA Isolation Kit (Roche Applied Science) according to the manufacturer's instuctions. RNA quantity and quality were assessed with a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies) and an Agilent Bioanalyzer 2100 with RNA 6000 LabChips (Agilent Technologies Netherlands BV).
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized with the Superscript III Reverse Transcriptase kit (Invitrogen) using 10 μg of total RNA as template and 360 U of SuperscriptTM III RT and purified with the Cyscribe GFX purification kit (Amersham Biosciences). The purified cDNA was labeled with Cy3- or Cy5-monoreactive dye and purified again with the Cyscribe GFX purification kit.
|
| |
|
| Channel 2 |
| Source name |
timepoint 37
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: strain 168
|
| Growth protocol |
Wild-type Bacillus subtilis 168 (trpC2; Anagnostopoulos and Spizizen, 1961) was grown in TY medium (10 g/L trypton, 5 g/L yeast extract, 5 g/L NaCl) in batch mode at 37°C in a 16 L fermentor using a working volume of 12 L. Cultures were aerated at 40 L/min and 400 rpm. The pH and oxygen levels were monitored, but were allowed to fluctuate during the batch culture incubation period. To prevent foaming, 0.3% antifoam A (SIGMA) was added on an as-needed basis. Cultures were inoculated to an OD600 of approximately 0.01 from synchronized exponentially grown pre-cultures grown at 37°C in TY and frozen in 10% glycerol (Lulko et al., 2007). Growth was monitored by measurement of the OD at 600 nm. As soon as the culture reached an OD600 of 0.1 (typically after around 75 min.), samples were taken every 10 minutes for optical density measurements and RNA isolation. In total 40 timepoints were sampled. Cells were harvested by centrifugation (14.000 rpm, 4°C, 1 min.) and the pellet was immediately frozen in liquid nitrogen and stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with the High Pure RNA Isolation Kit (Roche Applied Science) according to the manufacturer's instuctions. RNA quantity and quality were assessed with a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies) and an Agilent Bioanalyzer 2100 with RNA 6000 LabChips (Agilent Technologies Netherlands BV).
|
| Label |
Cy5
|
| Label protocol |
cDNA was synthesized with the Superscript III Reverse Transcriptase kit (Invitrogen) using 10 μg of total RNA as template and 360 U of SuperscriptTM III RT and purified with the Cyscribe GFX purification kit (Amersham Biosciences). The purified cDNA was labeled with Cy3- or Cy5-monoreactive dye and purified again with the Cyscribe GFX purification kit.
|
| |
|
| |
| Hybridization protocol |
The labeled cDNA was hybridized to oligonucleotide microarrays (Lulko et al., 2007) in Ambion Slidehyb #1 buffer (Ambion Europe Ltd) at 45°C for 16-18 h. After hybridization, slides were washed for 5 min at 37°C in 2x SSC with 0.5% SDS, 2x 5 min at 37°C in 1x SSC with 0.25% SDS and 1 min at 37°C in 1x SSC with 0.1% SDS, and then dried by centrifugation (2 min, 2000 rpm). SSC = 150 mM NaCl, 15 mM trisodium citrate.
|
| Scan protocol |
The microarrays were scanned with a GenePix Autoloader 4200AL confocal laser scanner (Molecular Devices Ltd). Determination of the individual intensities of each spot was done with ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, Md., USA) with a local corners background correction method.
|
| Description |
data 36 vs 37.slide #2 ratio
|
| Data processing |
Expression levels were processed and normalized (Lowess method) with MicroPrep (van Hijum et al., 2003). The median expression level was calculated for all genes based on the expression values from all six measurements for each timepoint. To facilitate the analysis of intensity levels of genes for each time point we divided the median intensity for each gene by the averaged intensity of all genes for each timepoint, which was followed by a natural log transformation. Normalized intensity of each signal (Cy3, Cy5) is given as well as the log2 ratio. The additional processed data file available on the Series record (TimepointProcessedData.txt) contains the natural log transformed median expression level all genes based on the expression values from all six measurements for each timepoint.
|
| |
|
| Submission date |
Jan 11, 2010 |
| Last update date |
Oct 31, 2011 |
| Contact name |
EJ Blom |
| E-mail(s) |
ejblom@gmail.com
|
| Organization name |
University of Groningen
|
| Department |
Molecular Genetics
|
| Street address |
Kerklaan 30
|
| City |
Haren |
| ZIP/Postal code |
9751 NN |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6031 |
| Series (1) |
| GSE19831 |
Growing pains: natural stress response during growth of B. subtilis (high density time-resolved transcriptome analysis) |
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