|
| Status |
Public on Mar 13, 2019 |
| Title |
Bacillus subtilis KMO grown in Mannos [Mannose.2.B] |
| Sample type |
RNA |
| |
|
| Source name |
Bacillus subtilis KMO grown in Mannos
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype/variation: KMO
growth media: Mannos
|
| Growth protocol |
Cells were grown to OD600 of 0.25 in GM17 media
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences
|
| |
|
| Hybridization protocol |
The protocol was performed as described in Hendriksen et al., 2008 (PMID: 18024519)
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner
|
| Description |
Bacillus subtilis KMO replicate 4, grown in Mannose
|
| Data processing |
Array images were analyzed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Prior to analysis, automatically and manually flagged spots and spots with very low background subtracted signal intensity (5% of the weakest spots ) were filtered out of all data sets.Net signal intensities were calculated by grid-based background subtraction. A grid-based Lowess transformation was performed for slide normalization, negative and empty values were removed, and outliers were removed by the deviation test. Expression ratios of mutant strain over the wild-type strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.
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| |
|
| Submission date |
Mar 12, 2019 |
| Last update date |
Mar 13, 2019 |
| Contact name |
Anne de Jong |
| E-mail(s) |
anne.de.jong@rug.nl
|
| Phone |
+31 50 363 2047
|
| Organization name |
university of Groningen
|
| Department |
Molecular Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL26118 |
| Series (1) |
| GSE128187 |
Phosphosugar stress in Bacillus subtilis: Intracellular accumulation of mannose 6-phosphate derepresed the glcR-phoC operon from repression by GlcR |
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