|
| Status |
Public on Oct 28, 2014 |
| Title |
Slide 347265 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cells
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype/variation: ulaR mutant
|
| Growth protocol |
Cells were grown in AM17 medium (M17 medium + 10mM ascorbic acid) and harvested at OD600 of 0.15.
|
| 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 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 a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences).
|
| |
|
| Channel 2 |
| Source name |
cells
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype/variation: Wild type
|
| Growth protocol |
Cells were grown in AM17 medium (M17 medium + 10mM ascorbic acid) and harvested at OD600 of 0.15.
|
| 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 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 Kloosterman et al., 2006 and Shafeeq et al., 2011
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner
|
| Description |
Sample 1
|
| Data processing |
Dual-channel array images were analyzed with Genepix 6. Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP software. 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 rokA 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 22, 2014 |
| Last update date |
Oct 28, 2014 |
| Contact name |
Sulman Shafeeq |
| E-mail(s) |
[email protected]
|
| Organization name |
Karolinska Institutet
|
| Department |
MTC
|
| Street address |
nobel vag 16
|
| City |
STOCKHOLM |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL11484 |
| Series (1) |
| GSE61649 |
Impact of ulaR delation on the transcriptome of Streptococcus pneumoniae D39 in the presence of ascorbic acid |
|
