|
| Status |
Public on Aug 04, 2015 |
| Title |
slide 176929 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Net{A}
|
| Organism |
Bacillus subtilis subsp. natto |
| Characteristics |
genotype: wild type
protocol: 0.7% agar
|
| Treatment protocol |
no treatment
|
| Growth protocol |
B subtilis Natto (wild type or spo0A derivative) was spotted LB agar plate (with 0.7 or 1.5% agar concentrations) and grown at 37C overnight. Cells were harvested from the agar plate and RNA purification was performed on the samples.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215). 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 NucleoSpin Extract II columns
|
| |
|
| Channel 2 |
| Source name |
Net{B}
|
| Organism |
Bacillus subtilis subsp. natto |
| Characteristics |
genotype: wild type
protocol: 1.5% agar
|
| Treatment protocol |
no treatment
|
| Growth protocol |
B subtilis Natto (wild type or spo0A derivative) was spotted LB agar plate (with 0.7 or 1.5% agar concentrations) and grown at 37C overnight. Cells were harvested from the agar plate and RNA purification was performed on the samples.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures.
|
| Label |
Cy5
|
| 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 NucleoSpin Extract II columns
|
| |
|
| |
| Hybridization protocol |
The protocol was performed as described in Lulko, et. al. J. Mol. Microbiol. Biotechnol. 12:82-95 PMID: 17183215
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner
|
| Description |
Sample 2
comparison of wild type at 1.5% and 0.7% agar concentration
|
| Data processing |
Dual-channel array images were analyzed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP as previously described (den Hengst et al J. Biol. Chem. 280:34332-34342; PMID: 16040604). Prior to analysis, automatically and manually flagged spots and spots with very low background subtracted signal intensity (5% of the weakest spots [sum of Cy3 and Cy5 net signals]) 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.
Net{A} is the raw signal of channel A minus the local corner background signal of channel A
Net{B} is the raw signal of channel B minus the local corner background signal of channel B
|
| |
|
| Submission date |
Jan 29, 2013 |
| Last update date |
Aug 04, 2015 |
| Contact name |
Akos T Kovacs |
| Organization name |
Friedrich Schiller University of Jena
|
| Department |
Institute of Microbiology
|
| Lab |
Terrestrial biofilms
|
| Street address |
Neugasse 23
|
| City |
Jena |
| ZIP/Postal code |
07743 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6031 |
| Series (1) |
| GSE43840 |
Sliding of Bacillus subtilis Natto |
|
