|
| Status |
Public on Jan 01, 2011 |
| Title |
JR32 strain grown to exponential phase (SCOTS treated), replicate 6 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
3XSCOTS cDNA obtained from JR32 grown to exponential phase
|
| Organism |
Legionella pneumophila subsp. pneumophila str. Philadelphia 1 |
| Characteristics |
media: AYE broth, 37C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol reagent (Gibco BRL) was used according to the manufacturer instruction. RNA was treated with DNase I (Ambion). RNA sample was converted to cDNA in 5 independent reverse-transcription reactions as described previously (Graham, J. 1999. PNAS 96:11554-559). Bacterial transcript were purified from host transcript by using 3 rounds of the SCOTS procedure (Daigle, F. 2002. Methods Enzymol. 358:108-122).
|
| Label |
AlexaFluor 546
|
| Label protocol |
2 µg of cDNA was labeled using amino-allyl dUTP (GE Healthcare) by random priming with Klenow (New England Biolabs) as previously described (Porwollik, S. 2001. Mutat Res 483:1-11.). Subsequently, the resulting cDNA was coupled to the succinimidyl ester fluorescent dye AlexaFluor 546.
|
| |
|
| Channel 2 |
| Source name |
Genomic DNA used as reference channel
|
| Organism |
Legionella pneumophila subsp. pneumophila str. Philadelphia 1 |
| Characteristics |
reference: genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Wizard Genomic Purification Kit (Promega).
|
| Label |
AlexaFluor 647
|
| Label protocol |
5 ug of genomic DNA was labeled with amino-allyl dUTP with klenow fragment and random hexamer as previously described (Porwollik, S. 2001. Mutat Res 483:1). Subsequently, the resulting DNA was coupled to the succinimidyl ester fluorescent dye AlexaFluor 647.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed as previously described (Hovel-Miner, G 2009. JB, 191:2461).
|
| Scan protocol |
Scanning and image acquisition was performed on a Scan Array Express (Perkin Elmer) using ScanArray 3.0 software as previously described (Hovel-Miner, G 2009. JB, 191:2461).
|
| Description |
L. pneumophila was grown to exponential phase in rich AYE broth. This is the control condition to identify L. pneumophila genes differentially expressed during intracellular multiplication.
|
| Data processing |
The data was background subtracted and normalized by calculating the contribution of each spot to the total intensity and the ratio to gDNA was recorded.
|
| |
|
| Submission date |
Jul 20, 2010 |
| Last update date |
Jan 01, 2011 |
| Contact name |
Sebastien Faucher |
| E-mail(s) |
[email protected]
|
| Phone |
514-398-7886
|
| Organization name |
McGill University
|
| Department |
Natural Resource Sciences
|
| Lab |
Faucher
|
| Street address |
21,111 Lakeshore
|
| City |
St-Anne-de-Bellevue |
| State/province |
QC |
| ZIP/Postal code |
H9X 3V9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7283 |
| Series (2) |
| GSE23029 |
Legionella pneumophila transcriptome during intracellular multiplication in human macrophages |
| GSE23032 |
Comparison of SCOTS and standard microarray protocol |
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