|
| Status |
Public on Nov 16, 2010 |
| Title |
1,500(1) CFU, t=15h, replicate 4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
GAF 1,500(1) CFU mutant library t=15h replicate 4
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: TIGR4 marinerT7 mutant library
mutant library size: 1,500(1) CFU
sample type: CSF
|
| Biomaterial provider |
Laboratory of Pediatric Infectious Diseases, RUNMC
|
| Treatment protocol |
Chromosomal DNA extracts was digested with AluI (New England Biolabs). The resulting DNA fragments were used as a template for an in vitro T7 RNA polymerase reaction (T7 MegaScript, Ambion). After removal of template DNA by DNAseI treatment, fluorescent Cy3/Cy5-labeled dUTP nucleotides (GE Healthcare) were incorporated by reverse transcription using Superscript III (Invitrogen).
|
| Growth protocol |
Control and CSF samples obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromosomal DNA was isolated from pneumococcal cultures by cetyl-trimethylammonium bromide (CTAB) extraction as described previously (PMID: 8057895).
|
| Label |
Cy3
|
| Label protocol |
Chromosomal DNA extracts was digested with AluI (New England Biolabs). The resulting DNA fragments were used as a template for an in vitro T7 RNA polymerase reaction (T7 MegaScript, Ambion). After removal of template DNA by DNAseI treatment, fluorescent Cy3/Cy5-labeled dUTP nucleotides (GE Healthcare) were incorporated by reverse transcription using Superscript III (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
GAF control 1,500(1) CFU mutant library
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: TIGR4 marinerT7 mutant library
mutant library size: 1,500(1) CFU
sample type: control
|
| Biomaterial provider |
Laboratory of Pediatric Infectious Diseases, RUNMC
|
| Treatment protocol |
Chromosomal DNA extracts was digested with AluI (New England Biolabs). The resulting DNA fragments were used as a template for an in vitro T7 RNA polymerase reaction (T7 MegaScript, Ambion). After removal of template DNA by DNAseI treatment, fluorescent Cy3/Cy5-labeled dUTP nucleotides (GE Healthcare) were incorporated by reverse transcription using Superscript III (Invitrogen).
|
| Growth protocol |
Control and CSF samples obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromosomal DNA was isolated from pneumococcal cultures by cetyl-trimethylammonium bromide (CTAB) extraction as described previously (PMID: 8057895).
|
| Label |
Cy5
|
| Label protocol |
Chromosomal DNA extracts was digested with AluI (New England Biolabs). The resulting DNA fragments were used as a template for an in vitro T7 RNA polymerase reaction (T7 MegaScript, Ambion). After removal of template DNA by DNAseI treatment, fluorescent Cy3/Cy5-labeled dUTP nucleotides (GE Healthcare) were incorporated by reverse transcription using Superscript III (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Fluorescently labeled DNA probes were combined and hybridized in Slidehyb buffer 1 (Ambion) to pneumococcal microarrays, which are super-amine glass slides spotted with duplicates of amplicons representing 2,087 S. pneumoniae TIGR4 ORFs and 184 ORFs unique to R6 (PMID: 16787930 ).The microarrays were incubated for 16 hours at 45°C, and then washed with 2xSSC (Invitrogen) containing 0.25% SDS for 5 min, followed by 2 washes in 1xSSC and 0.5xSSC for 5 min each. Finally, slides were dipped into H2O and dried by centrifugation.
|
| Scan protocol |
Dual channel array images were acquired on a GenePix 4200AL microarray scanner and analyzed with GenePix Pro software (Axon Instruments, Union City, CA, USA). Spots were screened visually to identify those of low quality.
|
| Description |
n.a.
|
| Data processing |
Microarray scan files were analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were screened visually to identify those of low quality and removed from the data set prior to analysis. A net mean intensity filter based on hybridization signals obtained with oligomer-spots representing open reading frames unique for S. pneumoniae strain R6 that were present on the pneumococcal microarray as well, was applied in all experiments. Slide data were processed and normalized using MicroPreP Slide data were processed and normalized using MicroPreP as described (PMID: 15130795). Further analysis was performed using a Cyber-T implementation of the Student’s t test (cybert.microarray.ics.uci.edu). This web-based program lists the ratios of all intra-replicates (duplicate spots) and inter-replicates (different slides), the mean ratios per gene, and standard deviations and (Bayesian) p values assigned to the mean ratios. For identification of conditionally essential genes, only genes with a minimum of 6/8 reliable measurements and a Bayesian p-value < 0.001 were included.
|
| |
|
| Submission date |
Apr 23, 2010 |
| Last update date |
Nov 16, 2010 |
| Contact name |
Peter Burghout |
| E-mail(s) |
[email protected]
|
| Phone |
+31-24-36666332
|
| Fax |
+31-24-3666352
|
| Organization name |
Radboud University Nijmegen Medical Centre
|
| Department |
Laboratory of Pediatric Infectious Diseases
|
| Street address |
Kapittelweg 29
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 EN |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6268 |
| Series (1) |
| GSE21729 |
Genome-wide identification of Streptococcus pneumoniae genes essential for the development of experimental meningitis |
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