|
| Status |
Public on May 25, 2016 |
| Title |
B. subtilis wild type_exponential growth_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pooled RNA (equal amounts of RNA from all samples)
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: Bacillus subtilis subsp. subtilis str. 168
|
| Treatment protocol |
Samples were taken at OD600 of 0.5.
|
| Growth protocol |
B. subtilis 168 wild type and gdpP pgpH double mutant cells were grown under vigorous agitation at 37 °C in Spizizen minimal medium supplemented with glutamate..
|
| 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, 5 µg of total RNA were mixed with random primers (FairPlay III Microarray Labeling Kit) 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, first-strand Master Mix, Actinomycin D
(final conc. 40 µg/ml) and AffinityScript HC Reverse Transcriptase were added. The reaction was incubated for 60 min at room temperature and for 60 min at 42°C. After hydrolyzing the RNA, cDNA was precipitated overnight at -20°C. NHS-ester dye coupling (CyDye Mono-Reactive Dye,
GE Healthcare) and purification of labeled cDNA were performed according to the FairPlay III instructions. cDNA and Cy-dye concentrations were quantified by means of a NanoDrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
B. subtilis wild type_exponential growth
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: Bacillus subtilis subsp. subtilis str. 168
genotype/variation: wild type
|
| Treatment protocol |
Samples were taken at OD600 of 0.5.
|
| Growth protocol |
B. subtilis 168 wild type and gdpP pgpH double mutant cells were grown under vigorous agitation at 37 °C in Spizizen minimal medium supplemented with glutamate..
|
| 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 |
Cy5
|
| Label protocol |
For cDNA synthesis, 5 µg of total RNA were mixed with random primers (FairPlay III Microarray Labeling Kit) 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, first-strand Master Mix, Actinomycin D
(final conc. 40 µg/ml) and AffinityScript HC Reverse Transcriptase were added. The reaction was incubated for 60 min at room temperature and for 60 min at 42°C. After hydrolyzing the RNA, cDNA was precipitated overnight at -20°C. NHS-ester dye coupling (CyDye Mono-Reactive Dye,
GE Healthcare) and purification of labeled cDNA were performed according to the FairPlay III instructions. cDNA and Cy-dye concentrations were quantified by means of a NanoDrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
100 ng of Cy5-labeled cDNA and 100 ng of Cy3-labeled cDNAwere 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 |
wt_3
|
| Data processing |
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies).
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 |
Mar 30, 2016 |
| Last update date |
May 25, 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 |
GPL21669 |
| Series (1) |
| GSE79716 |
Transcriptome changes in response to accumulation of cyclic di-AMP in Bacillus subtilis. |
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