|
| Status |
Public on Jan 11, 2010 |
| Title |
DeltaREL vs WT_20min_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S. pneumoniae D39 mutant (IU1921), untreated
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: D39 (NCTC 7466)
serotype: 2
genotype: relSpn gene (spd1458) is deleted
|
| Biomaterial provider |
Winkler laboratory stock
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Bacteria were grown statically in Brain Heart Infusion media (Bacto BHI, Becton Dickinson) at 37C in an atmosphere of 5% CO2. Culture density OD620~0.1 is defined as (t=0 min)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted at (t=20 min) using a hot lysis/ acid phenol procedure followed by on-column DNase treatment and purification (RNAeasy mini kit, Qiagen) as described in Robertson et al., J Bact (2002) 184:3508-3520
|
| Label |
Cy5
|
| Label protocol |
Single strand cDNA synthesis and direct labeling performed using 30 micrograms of total RNA and Superscript II reverse transcriptase (Invitrogen) as per microarray manufacturer's recommendations (http://www.ocimumbio.com/web/arrays/assets/downloads/manuals/manual_bacteria.pdf)
|
| |
|
| Channel 2 |
| Source name |
S. pneumoniae D39 (IU1690), untreated
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: D39 (NCTC 7466)
serotype: 2
genotype: wildtype
|
| Biomaterial provider |
Winkler laboratory stock of NCTC 7466 (Health Protection Agency, London, UK)
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Bacteria were grown statically in Brain Heart Infusion media (Bacto BHI, Becton Dickinson) at 37C in an atmosphere of 5% CO2. Culture density OD620~0.1 is defined as (t=0 min)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted at (t=20 min) using a hot lysis/ acid phenol procedure followed by on-column DNase treatment and purification (RNAeasy mini kit, Qiagen) as described in Robertson et al., J Bact (2002) 184:3508-3520
|
| Label |
Cy3
|
| Label protocol |
Single strand cDNA synthesis and direct labeling performed using 30 micrograms of total RNA and Superscript II reverse transcriptase (Invitrogen) as per microarray manufacturer's recommendations (http://www.ocimumbio.com/web/arrays/assets/downloads/manuals/manual_bacteria.pdf)
|
| |
|
| |
| Hybridization protocol |
Fluorescently-labeled cDNAs were resuspended in the manufacturer's salt-based hybridization buffer. Samples were heated for 3 min at 95C, chilled on ice for 3 min, and applied to the microarray slide. Slides were hybridized at 42C overnight (~18 hrs). Slides were washed for 5 min in prewarmed (30C) buffers. Buffer 1: 2X SSC, 0.1% SDS; Buffer 2: 1X SSC; Buffer 3: 0.1X SSC.
|
| Scan protocol |
Microarray slides were scanned using an Axon GenePix 4200A scanner at gain settings which balanced the channel signals (ratio~0.1). Images were analyzed using GenePix Pro 5.0 software (Molecular Devices).
|
| Description |
Independent biological replicate, dye swap
|
| Data processing |
Spots flagged as bad in more than one replicate were removed from further analysis. The Bioconductor marray package was used to perform loess normalization on background-subtracted data. Ribosomal proteins were excluded from the normalization procedure. See associated publication (Kazmierczak, KM et al., Roles of relSpn in Stringent Response, Global Regulation, and Virulence of Serotype 2 Streptococcus pneumoniae D39, submitted) for further details
|
| |
|
| Submission date |
Jan 23, 2009 |
| Last update date |
Jan 11, 2010 |
| Contact name |
Krystyna M Kazmierczak |
| Organization name |
Indiana University
|
| Department |
Biology
|
| Street address |
1001 E. Third St.
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405-3700 |
| Country |
USA |
| |
|
| Platform ID |
GPL536 |
| Series (1) |
| GSE14750 |
Roles of relSpn in Stringent Response, Global Regulation, and Virulence of Serotype 2 Streptococcus pneumoniae D39 |
|
