|
| Status |
Public on Nov 20, 2007 |
| Title |
codY S.pneu TIGR4 164687 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
dcodY OD 0.2
|
| Organism |
Streptococcus pneumoniae TIGR4 |
| Characteristics |
prep 5
|
| Extracted molecule |
total RNA |
| Extraction protocol |
500 ml of THY-broth was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37°C. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of either 0.1 or 0.2 (early and mid-log growth, respectively). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (PMID: 17085554). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (PMID: 17085554 and 15907200). In all cases, dye-swapping was performed with one of the three biological replicates.
|
| Label |
cy3
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions
|
| |
|
| Channel 2 |
| Source name |
wt OD 0.2
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
prep 6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
500 ml of THY-broth was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37°C. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of either 0.1 or 0.2 (early and mid-log growth, respectively). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (PMID: 17085554). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (PMID: 17085554 and 15907200). In all cases, dye-swapping was performed with one of the three biological replicates.
|
| Label |
cy5
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions
|
| |
|
| |
| Hybridization protocol |
Hybridization (16 h at 45 °C) of labeled cDNA was performed in Ambion Slidehyb #1 hybridization buffer (Ambion Europe) on superamine glass slides (Array-It; SMMBC), containing technical replicates.
|
| Scan protocol |
Slides were scanned using the Genepix 4200AL DNA micro array slide scanner.
Quantification of the signal of the spots on slides was done with ArrayPro 4.5.
|
| Description |
codY-mutant of Streptococcus pneumoniae versus Wild type
|
| Data processing |
Slide data were processed and normalized using MicroPreP, and CyberT implementation of the Student's t test (PMID: 15130795).
|
| |
|
| Submission date |
Mar 23, 2007 |
| Last update date |
Nov 20, 2007 |
| 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 |
GPL5023 |
| Series (1) |
| GSE7350 |
CodY of Streptococcus pneumoniae: link between nutritional gene regulation and virulence |
|
