|
| Status |
Public on Jul 31, 2019 |
| Title |
Slide 344251 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
F635 Mean
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype: Wild Type
|
| Growth protocol |
Cells were grown to OD600 of 0.3 in GM17 medium
|
| 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; Shafeeq et al., 2011). 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 |
F532 Mean
|
| Organism |
Streptococcus pneumoniae D39 |
| Characteristics |
genotype: plcR Mutant
|
| Growth protocol |
Cells were grown to OD600 of 0.3 in GM17 medium
|
| 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; Shafeeq et al., 2011). 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.
|
| Scan protocol |
Scanning was done using the Genepix 4200AL laser scanner.
|
| Description |
Avarage ratio of inter slide replicates
|
| Data processing |
Dual-channel array images were analyzed with Genpix software. Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP as previously described (15, 18, 49). 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 WT over mutant strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.
|
| |
|
| Submission date |
Jun 19, 2019 |
| Last update date |
Aug 02, 2019 |
| 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) |
| GSE133010 |
D39 wild-type vs D39 ΔplcR in GM17 medium |
|
