|
| Status |
Public on May 18, 2020 |
| Title |
B. subtilis_SMM-control_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
B. subtilis_ SMM_OD1.0
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: BSB1 (Trp+ derivative of strain 168)
medium: SMM control
|
| Growth protocol |
B. subtilis BSB1 was grown aerobically at 37 °C in Spizizen's minimal medium either supplemented with 1 mM glycine betaine, 1.2 M NaCl or 1.2 M NaCl and 1 mM glycine betaine. Cells were harvested at OD at 600 nm of 1.0.
|
| 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 (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 |
1200 ng of Cy3-labeled cDNA were hybridized to the tiling array following Agilent’s hybridization, washing and scanning protocol (One-Color Microarray-based Gene Expression Analysis, version 5.5).
|
| Scan protocol |
The microarray was scanned using an Agilent Microarray Scanner G2505C (Agilent Technologies).
|
| Description |
SMM-control_3
Bacillus subtilis BSB1
|
| Data processing |
Data were extracted using the Agilent Feature Extraction software (version 11.5.1.1, Agilent Technologies). Probe intensities were computed from the raw intensity data using a model of signal shift and drift and correcting for probe affinity variations as described before (Nicolas et al., 2009; PMID 19561016). An aggregated expression value was computed for each annotated CDS and previously identified RNA feature (Nicolas et al., 2012; PMID 22383849) as the median log2 intensity of probes lying entirely within the corresponding region.
|
| |
|
| Submission date |
Feb 11, 2020 |
| Last update date |
May 19, 2020 |
| 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 |
GPL21981 |
| Series (1) |
| GSE145124 |
Impact of high salinity and the compatible solute glycine betaine on global gene expression of Bacillus subtilis |
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