|
| Status |
Public on Aug 31, 2012 |
| Title |
RM6607W-Stationary phase-REP3-3 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Escherichia coli RM6607W-Stationary phase
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: Escherichia coli O157:H7 str. RM6607W
|
| Growth protocol |
Liquid cultures of E. coli were obtained by inoculation of colonies into LB broth and growth at 37°C with shaking (150 rpm) to stationary phase (16 h).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
E. coli genomic DNA was prepared with the Qiagen Dneasy Blood and Tissue kit. Total E. coli RNA was extracted from stationary phase cultures with the Promega SV Total RNA Isolation Kit according to the manufacturer’s specifications.
|
| Label |
Cy5
|
| Label protocol |
2 µg of genomic DNA were Cy3-dUTP labeled during random primed synthesis with Klenow fragment (NEB) at 37°C for 16 h. 20 µg of total RNA were labeled during reverse transcription with random primed hexamers to cDNA with Cy5-dUTP using Stratascript (Stratagene, Palo Alto, CA) at 42°C for 16 h.
|
| |
|
| Channel 2 |
| Source name |
Genomic DNA E. coli strain RM6607
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: Escherichia coli O157:H7 str. RM6607
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
E. coli genomic DNA was prepared with the Qiagen Dneasy Blood and Tissue kit. Total E. coli RNA was extracted from stationary phase cultures with the Promega SV Total RNA Isolation Kit according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
2 µg of genomic DNA were Cy3-dUTP labeled during random primed synthesis with Klenow fragment (NEB) at 37°C for 16 h. 20 µg of total RNA were labeled during reverse transcription with random primed hexamers to cDNA with Cy5-dUTP using Stratascript (Stratagene, Palo Alto, CA) at 42°C for 16 h.
|
| |
|
| |
| Hybridization protocol |
Labeled reference genomic DNA and test cDNAs were combined in 45 µl Pronto! cDNA hybridization solution (Corning, Corning, NY) and heated to 95°C for 5 min. Then, 15 µl of the hybridization mixture was put onto a microarray slide and sealed with a cover slip. The microarray slide was placed in a hybridization chamber (Corning) and incubated at 42°C for 18 h. Following hybridization, the slides were washed twice in 2× SSC, 0.1% sodium dodecyl sulfate at 42°C for 10 min, followed by twice in 1× SSC at room temperature for 10 min, and finally twice in 0.2× SSC at room temperature for 5 min. The microarray slides were dried by centrifugation at 300 × g for 10 min before scanning.
|
| Scan protocol |
Scanned on an Axon GenePix4000B scanner at 10nm resolution using GenePix software.
Images were quantified using GenePix software (version 6.0).
|
| Data processing |
Fluorescence ratios were calculated after local background was subtracted from spot signals. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization (centering) was performed by bringing the median natural logarithm of the ratios for each group of spots printed by the same pin to zero using the following equation: ln(Ti ) = ln(Ri /Gi ) - c, where T is the centered ratio, i is the gene index, R and G are the red and green intensities, respectively, and c is the 50th percentile of all red/green ratios.
|
| |
|
| Submission date |
Jul 17, 2012 |
| Last update date |
Aug 31, 2012 |
| Contact name |
Craig T Parker |
| E-mail(s) |
CRAIG.PARKER@ARS.USDA.GOV
|
| Phone |
510-559-6187
|
| Organization name |
USDA ARS
|
| Department |
PSM
|
| Street address |
800 Buchanan St
|
| City |
Albany |
| State/province |
CA |
| ZIP/Postal code |
94710 |
| Country |
USA |
| |
|
| Platform ID |
GPL14768 |
| Series (1) |
| GSE39439 |
RcsB contributes to the distinct stress fitness between Escherichia coli O157:H7 curli variants of 1993 Hamburger-associated outbreak strains |
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