|
| Status |
Public on Oct 01, 2011 |
| Title |
186051_gal_M_wtCy5-ccpACy3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Net intensity (tr.mean) {A}
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype/variation: Wild type
|
| Growth protocol |
Cells were grown to MID-log phase in CDM medium with Galactose as the sole carbon source
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Kloosterman et al., 2006). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from Four 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 a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences
|
| |
|
| Channel 2 |
| Source name |
Net intensity (tr.mean) {B}
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype/variation: ccpA mutant
|
| Growth protocol |
Cells were grown to MID-log phase in CDM medium with Galactose as the sole carbon source
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Kloosterman et al., 2006). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from Four 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 Kloosterman TG et al., 2006b
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner
|
| Description |
Sample 1
|
| 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. 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 copY mutant strain over the D39 wild-type strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.
|
| |
|
| Submission date |
Sep 01, 2011 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Anne de Jong |
| E-mail(s) |
[email protected]
|
| 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 |
GPL11484 |
| Series (2) |
| GSE31817 |
ccpA mutant compared to D39 wild-type in Streptococcus pneumoniae in CDM + Galactose at MID-log growth phase |
| GSE31819 |
CcpA Ensures Optimal Metabolic Fitness of Streptococcus pneumoniae |
|
