|
| Status |
Public on Mar 31, 2014 |
| Title |
B. subtilis_glucose limitation_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pooled RNA (equal amounts of RNA from all samples)
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: BSB1 (Trp+ derivative of strain 168)
|
| Treatment protocol |
The medium was supplemented with 70.13 g/l NaCl (1.2 M) with or without 117.15 mg/l glycine betaine (1 mM) to mimic osmotic stress and osmoprotection of B. subtilis cells by an externally provided compatible solute. Cultivations were performed until metabolic steady state was reached, i.e. for five volume changes.
|
| Growth protocol |
B. subtilis BSB1 was grown in minimal M9 medium with 1 g/l glucose as sole carbon source. Continuous cultivation was performed at 0.1 h-1 in a parallelized 1 l bioreactor system with a working volume of 300 ml, kept at an aeration rate of 9 l/h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002).
|
| Label |
Cy3
|
| Label protocol |
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies). The RNA/primer mixture was incubated at 70 °C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare) and 2µl of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 60 min and then heated to 70 °C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
B. subtilis wild type_glucose lim. chemostat
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: BSB1 (Trp+ derivative of strain 168)
|
| Treatment protocol |
The medium was supplemented with 70.13 g/l NaCl (1.2 M) with or without 117.15 mg/l glycine betaine (1 mM) to mimic osmotic stress and osmoprotection of B. subtilis cells by an externally provided compatible solute. Cultivations were performed until metabolic steady state was reached, i.e. for five volume changes.
|
| Growth protocol |
B. subtilis BSB1 was grown in minimal M9 medium with 1 g/l glucose as sole carbon source. Continuous cultivation was performed at 0.1 h-1 in a parallelized 1 l bioreactor system with a working volume of 300 ml, kept at an aeration rate of 9 l/h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002).
|
| Label |
Cy5
|
| Label protocol |
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies). The RNA/primer mixture was incubated at 70 °C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare) and 2µl of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 60 min and then heated to 70 °C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
600 ng of Cy5-labeled cDNA and 600 ng of Cy3-labeled cDNA were hybridized to the microarray following Agilent’s hybridization, washing and scanning protocol (Two-Color Microarray-based Gene Expression Analysis, version 5.5).
|
| Scan protocol |
The microarray was scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies).
|
| Data processing |
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies). The normalization/ dye bias correction method is: Linear and Lowess.
The normalization/ dye bias correction method as defined by the Agilent Feature Extraction protocol for 2 color gene expression arrays (protocol GE2_105_Dec08) is: Linear and Lowess. The normalization/ dye bias correction method is: Linear and Lowess.
|
| |
|
| Submission date |
Dec 15, 2013 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Ulrike Mäder |
| E-mail(s) |
ulrike.maeder@uni-greifswald.de
|
| 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 |
GPL10901 |
| Series (1) |
| GSE53333 |
Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge |
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