|
| Status |
Public on Apr 18, 2016 |
| Title |
B. subtilis_M9SE medium_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
B. subtilis wild type_exponential growth in M9SE medium
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: Bacillus subtilis BSB1 (Trp+ derivative of strain 168)
|
| Growth protocol |
B. subtilis BSB1 was grown aerobically at 37 °C in S medium, CH medium, CHG medium (Sharpe et al., 1998; PMID 9457856) or M9SE medium (M9 succinate/glutamate) (PMID 22383849).
|
| 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; PMID 11948165).
|
| Label |
Cy3
|
| Label protocol |
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (Promega) and spike-ins (One-Color RNA Spike-In Kit, Agilent Technologies) and 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, 5 µl of 0.1 M DTT, 0.5 µl of dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP (GE Healthcare) and 2 µl of SuperScript II reverse transcriptase (Invitrogen). The reaction 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 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 G2505C (Agilent Technologies).
|
| Description |
growth rate: μ=0.4 h-1
|
| Data processing |
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies).
|
| |
|
| Submission date |
Feb 19, 2016 |
| Last update date |
Apr 19, 2016 |
| 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) |
| GSE78108 |
Growth-rate-dependent gene expression in Bacillus subtilis |
|
