|
| Status |
Public on Sep 02, 2016 |
| Title |
WT81-176-1-1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
strain WT81-176
|
| Organism |
Campylobacter jejuni |
| Characteristics |
genotype/variation: wild-type
|
| Growth protocol |
Campylobacter were grown under micro-aerobic conditions (5% O2, 10% CO2, and 85% N2) at 42°C on blood agar base II medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Campylobacter genomic DNA was prepared with the Qiagen Dneasy Blood and Tissue kit. To isolate total RNA, log phase cultures suspended in 10X stop solution (95% ethanol plus 5% phenol), incubated on ice for 20 min, pelleted by centrifugation. Pellets were suspended in 1 mL of RiboZol (Amresco) and RNA was removed by chloroform extraction. Total RNA (60 μg) was treated with DNaseI (Invitrogen) and then purified through an RNeasy Mini Column (Qiagen) according to the manufacturer’s specifications.
|
| Label |
Cy5
|
| Label protocol |
2 µg of genomic DNA were Cy5-dUTP labeled during random primed synthesis with Klenow fragment. 20 µg of total RNA were labeled during reverse transcription with random primed hexamers to cDNA with Cy3-dUTP using Stratascript (Stratagene, Palo Alto, CA) at 42°C for 16 h.
|
| |
|
| Channel 2 |
| Source name |
strain WT81-176
|
| Organism |
Campylobacter jejuni |
| Characteristics |
genotype/variation: wild-type
|
| Growth protocol |
Precultures of Campylobacter were grown under micro-aerobic conditions (5% O2, 10% CO2, and 85% N2) at 42°C in Heart Infusion broth (HI) , diluted to an A550 of 0.02 in 5 ml of HI, and incubated on a gyratory shaker (150 rpm) under microaerobic conditions at 42°C to stationary phase (A550 of 1.0-1.4) .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Campylobacter genomic DNA was prepared with the Qiagen Dneasy Blood and Tissue kit. To isolate total RNA, log phase cultures suspended in 10X stop solution (95% ethanol plus 5% phenol), incubated on ice for 20 min, pelleted by centrifugation. Pellets were suspended in 1 mL of RiboZol (Amresco) and RNA was removed by chloroform extraction. Total RNA (60 μg) was treated with DNaseI (Invitrogen) and then purified through an RNeasy Mini Column (Qiagen) according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
2 µg of genomic DNA were Cy5-dUTP labeled during random primed synthesis with Klenow fragment. 20 µg of total RNA were labeled during reverse transcription with random primed hexamers to cDNA with Cy3-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. Optimize intensities manually.
Images were quantified using GenePix software (version 6.0).
|
| Description |
C. jejuni strain 81-176 wildtype Bio Replicate 1 Tech 1
|
| 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.
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| |
|
| Submission date |
Sep 01, 2016 |
| Last update date |
Sep 02, 2016 |
| 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 |
GPL19897 |
| Series (1) |
| GSE86327 |
Microbiota-derived short-chain fatty acids modulate expression of Campylobacter jejuni determinants required for commensalism and virulence |
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