|
| Status |
Public on Dec 01, 2009 |
| Title |
B. subtilis_salt stress_120min_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pooled RNA (equal amounts of RNA from all samples)
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: wild type
reference: pooled RNA (equal amounts of RNA from all samples)
|
| Treatment protocol |
Exponentially growing B. subtilis cells (OD500nm of 0.4) were challenged with 6% (w/v) NaCl and samples were taken before and 10, 30, 60 and 120 minutes subsequent to the addition of NaCl.
|
| Growth protocol |
The B. subtilis 168 wild type strain was grown aerobically at 37 °C in BMM medium (Stülke et al., 1993) lacking glutamate to avoid its usage as precursor for biosynthesis of the compatible solute proline.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002).
|
| Label |
Cy3
|
| Label protocol |
For cDNA synthesis, 25 µg of total RNA were mixed with a B. subtilis gene specific primer mix (final concentration of 2.5 nM) (Eurogentec). As an external control, 1 ng of Arabidopsis thaliana rbcL RNA (Stratagene) was added. The RNA/primer mixture was incubated at 65 °C for 10 min followed by 10 min incubation at room temperature. Then, the following reagents were added: 10 µL of 5 x First Strand Buffer, 5 µl of 0.1 M DTT, 4 µL of dNTP mix (5 mM dATP; 5 mM dCTP; 5 mM dGTP; 2 mM dTTP) (Roche Diagnostics), 0.1 mM Cy3-dUTP or Cy5 dUTP (GE Healthcare), 1 µL SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 2 h. Subsequently, the RNA was hydrolyzed by incubation with 10 µL of 1M NaOH at 65 °C for 10 min. After adding 10 µL of 1M HCl and 200 µL of TE buffer (pH 8.0), the labeled cDNA was purified using the CyScribe GFX Purification Kit (GE Healthcare). After purification both samples were combined and the volume was reduced in a vacuum centrifuge. The labeled cDNA mixture was resuspended in 17 µL of hybridization buffer (MWG AG Biotech) and hybridized to the microarray.
|
| |
|
| Channel 2 |
| Source name |
salt stressed cells - 120 min after addition of 6% NaCl
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: wild type
stress: salt
time point: 120 min
|
| Treatment protocol |
Exponentially growing B. subtilis cells (OD500nm of 0.4) were challenged with 6% (w/v) NaCl and samples were taken before and 10, 30, 60 and 120 minutes subsequent to the addition of NaCl.
|
| Growth protocol |
The B. subtilis 168 wild type strain was grown aerobically at 37 °C in BMM medium (Stülke et al., 1993) lacking glutamate to avoid its usage as precursor for biosynthesis of the compatible solute proline.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002).
|
| Label |
Cy5
|
| Label protocol |
For cDNA synthesis, 25 µg of total RNA were mixed with a B. subtilis gene specific primer mix (final concentration of 2.5 nM) (Eurogentec). As an external control, 1 ng of Arabidopsis thaliana rbcL RNA (Stratagene) was added. The RNA/primer mixture was incubated at 65 °C for 10 min followed by 10 min incubation at room temperature. Then, the following reagents were added: 10 µL of 5 x First Strand Buffer, 5 µl of 0.1 M DTT, 4 µL of dNTP mix (5 mM dATP; 5 mM dCTP; 5 mM dGTP; 2 mM dTTP) (Roche Diagnostics), 0.1 mM Cy3-dUTP or Cy5 dUTP (GE Healthcare), 1 µL SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 2 h. Subsequently, the RNA was hydrolyzed by incubation with 10 µL of 1M NaOH at 65 °C for 10 min. After adding 10 µL of 1M HCl and 200 µL of TE buffer (pH 8.0), the labeled cDNA was purified using the CyScribe GFX Purification Kit (GE Healthcare). After purification both samples were combined and the volume was reduced in a vacuum centrifuge. The labeled cDNA mixture was resuspended in 17 µL of hybridization buffer (MWG AG Biotech) and hybridized to the microarray.
|
| |
|
| |
| Hybridization protocol |
The microarray was prehybridized at 42 °C for 45 min in 4 x SSC, 0.5% SDS, 1% BSA. Afterwards, the slide was washed six times with deionized water and dried at 1,500 rpm for 5 min. Hybridization was performed using a SlideBooster (Advalytix). The labeled cDNA mixture was denatured for 2 min at 95 °C and then hybridized to the microarray for 16 h at 42 °C. Subsequently, the slide was washed at room temperature for 5 min in 0.2 x SSC, 0.1% SDS and two times for 5 min in 0.2 x SSC and then dried at 1,500 rpm for 5 min.
|
| Scan protocol |
The microarray was scanned using a ScanArray Express scanner (PerkinElmer Life and Analytical Sciences). Data were extracted using the ScanArray Express image analysis software. Intensity values for Cy3 and Cy5 (feature pixel median minus background pixel median) were used for further analysis.
|
| Description |
B. subtilis grown in minimal medium
|
| Data processing |
Intensity-dependent (Lowess) normalization was performed using the GeneSpring software (Agilent Technologies). The Cy5/Cy3 ratios of duplicate spots were averaged resulting in one value per gene. These values represent the ratio of an individual sample/ common reference pool.
|
| |
|
| Submission date |
Sep 30, 2009 |
| Last update date |
Sep 30, 2009 |
| Contact name |
Ulrike Mäder |
| E-mail(s) |
[email protected]
|
| Organization name |
University Medicine Greifswald
|
| Department |
Functional Genomics
|
| Street address |
F.-L.-Jahn-Str. 15A
|
| City |
Greifswald |
| ZIP/Postal code |
D-17489 |
| Country |
Germany |
| |
|
| Platform ID |
GPL3502 |
| Series (1) |
| GSE18345 |
Transcriptome analysis of Bacillus subtilis salt stress adaptation |
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