|
| Status |
Public on Mar 01, 2018 |
| Title |
345429_D39PcpsT=>CCy3vsD39Cy5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
medium without uracil
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: D39PcpsT=>C
medium: without uracil
|
| Growth protocol |
Cells were grown to MID-log phase in CDM without uracil
|
| 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)
|
| |
|
| Channel 2 |
| Source name |
medium without uracil
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: D39
medium: without uracil
|
| Growth protocol |
Cells were grown to MID-log phase in CDM without uracil
|
| 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)
|
| |
|
| |
| 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 Genepix Pro software. 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 mutant strain and the D39 wild-type strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.
|
| |
|
| Submission date |
Jan 12, 2018 |
| Last update date |
Mar 01, 2018 |
| Contact name |
Tomas Kloosterman |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Groningen
|
| Department |
Natural Sciences
|
| Lab |
Molecular Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11484 |
| Series (2) |
| GSE109124 |
Comparing transcriptome by means of DNA microarrays of Streptococcus pneumoniae D39 versus D39PcpsT=>C in medium without uracil |
| GSE109129 |
Interplay between capsule expression and uracil metabolism in Streptococcus pneumoniae |
|
