|
| Status |
Public on Oct 09, 2012 |
| Title |
V. campbellii, dark, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
ATCC BAA-1116 dark
|
| Organism |
Vibrio campbellii CAIM 519 = NBRC 15631 = ATCC 25920 |
| Characteristics |
strain: BAA-1116
treatment: dark
|
| Treatment protocol |
Bacterial cultures were exposed to 24 hours of continuous dark or a 12 hour dark:12 hour light cycle.
|
| Growth protocol |
Ten total cultures (five biological replicates of the wild type (STR) V. campbellii BAA-1116 grown under continuous dark for 24 hours and five biological replicates of the wild type (STR) V. campbellii BAA-1116 grown under a 12 hour dark:12 hour light cycle [white light illumination at 54 µmol photons s-1 m-2]) were seeded with 5.0E+5 bacteria/mL in 25 mL M9 minimal salts medium plus glucose in 125 mL baffled Erlenmeyer flasksand. Cultures were incubated at 30°C and 200 rpm for 24 hours and then harvested for total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RiboPure Bacteria Kit (Ambion Inc., Austin, TX) according to the manufacturer's instructions. The quality and quantity of RNA samples were determined using the RNA 6000 Nano kit with 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) and NanoDrop ND2000 (Thermo Scientific Inc., Waltham, MA) spectrophotometer. The RNA samples were accepted for mRNA enrichment if the A260/A280 ratios were between 1.9-2.1, the ratio of intensities of the 23S and 16S rRNA were 1.5-2.5: 1 and the RNA integrity no. was between 7 and 10. mRNA enrichment was performed with 10 ug total RNA using using the MICROBExpress kit (Ambion) according to the manufacturer’s protocol. The quantity of mRNA samples were determined by NanoDrop ND2000 (Thermo Scientific Inc., Waltham, MA) spectrophotometer.
|
| Label |
biotin
|
| Label protocol |
Target preparation and hybridization to arrays: 600 ng of mRNA contained PolyA RNA controls (Affymetrix, Santa Clara, CA) with known concentrations to monitor the quality of the procedures. The labeling reaction was carried out in 30 uL reaction volume using KREATECH’s aRNA Labeling Kit (KREATECH Biotechnology, Amsterdam, the Netherlands) according to the manufacture’s recommended protocol. Labeled mRNAs were purified and fragmented using fragmentation buffer from Ambion (Austin, TX). The fragmentation from the reaction was carried out in a volume of 33 uL containing 1X fragmentation buffer (Ambion), labeled mRNA, and incubated at 70°C for 15 minutes. After incubation, the reaction was stopped by adding 1 uL of stop solution.
|
| |
|
| Hybridization protocol |
The cocktail was hybridized onto the custom designed Vharveyi520694 microarray (Affymetrix). Hybridization was carried out for 16 h at 49°C with rotation at 60 rpm. The microarrays were then washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the GeneChip 7G Scanner.
|
| Scan protocol |
The microarrays were then washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the GeneChip Scanner 3000. GeneChip® Operating Software 1.2 (GCOS 1.2) (Affymetrix) was used to acquire and process array images, determine the quantity and presence or absence of a transcript using the Microarray Suite 5.0 (MAS5) algorithm, and generated the Report files summarizing the quality of target detection for each microarray.
|
| Description |
Gene expression data from wild type V. campbellii (STR) grown for 24 hours in the dark
|
| Data processing |
CEL files were processed using the Bioconductor affy package.
|
| |
|
| Submission date |
Feb 10, 2012 |
| Last update date |
Oct 09, 2012 |
| Contact name |
Baochuan Lin |
| E-mail(s) |
[email protected]
|
| Phone |
202-767-0289
|
| Organization name |
US Naval Research Laboratory
|
| Department |
Center for Bio/Molecular Science & Engineering
|
| Street address |
4555 Overlook Avenue, S. W.
|
| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20375 |
| Country |
USA |
| |
|
| Platform ID |
GPL10230 |
| Series (1) |
| GSE35702 |
Photoinduction of the Vibrio campbellii Type III Secretion System |
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