|
| Status |
Public on Jun 01, 2010 |
| Title |
V campbellii, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
ATCC-BAA1116 V. campbellii at mid-log phase
|
| Organism |
Vibrio campbellii CAIM 519 = NBRC 15631 = ATCC 25920 |
| Characteristics |
growth phase: Mid-log phase
|
| Treatment protocol |
No treatment
|
| Growth protocol |
V. campbellii were grown in 50 mL polypropylene centrifuge tubes containing 5 mL of Luria Marine media and were incubated at 30°C for 12 hours with 250 rpm agitation until it reach cell density at Mid-log phase, 1.0E+8 to 1.0E+10 cells. Diploid strains MLY61 a/alpha (ura3-52/ura3-52 MATa/alpha) and MLY132 a/alpha (delta gpa2::G418/delta gpa2::G418 ura3-52/ura3-52 MATa/alpha) were obtained from J. Heitman, Duke University{Lorenz, 2000 #19; Lorenz, 1997 #33}. Standard YPM media were used as described{Sherman, 1991 #36}. Synthetic low ammonium media consisting of SLAM (0.17% Yeast Nitrogen Base without ammonium sulfate or amino acids, 50 µM ammonium sulfate, 2% maltose) were used when switching to low nitrogen pseudohyphal inducing conditions{Lorenz, 2000 #19; Lorenz, 1997 #33}. Similar to a previously described method{Pan, 2000 #37}, strains were grown to an OD600 of 1.0 in YPM media. Approximately 200 OD600 units were collected by filtering followed by resuspension in 10 mL RNAlater (Ambion) for <30 min, pelleted by centrifugation, flash frozen and stored at –80 °C. Strains were also grown to an OD600 of 1.0 in YPM media and approximately 200 O.D. were collected by filtration and transferred to SLAM media for 2 h at 30 °C prior to collection as above.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RiboPure Bacteria Kit (Ambion Inc., Austin, TX) according to the manufacturer's instructions with adjustment of the volume of ethanol (1.25 instead of 0.5) added to the aqueous phase to capture small RNA. mRNA enrichment were 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 ND1000 (Thermo Scientific Inc., Waltham, MA) spectrophotometer.
|
| Label |
Biotin
|
| Label protocol |
Target Preparation and Hybridization to Arrays: 1 µg of mRNA contained PolyA RNA controls (Affymetrix, Santa Clara, CA) with known concentrations to monitor the quality of the procedures . The labeling reaction is 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). Fragmentation reaction was carried out in a reaction volume of 33 uL using fragmentation buffer from Ambion.
|
| |
|
| Hybridization protocol |
The cocktail was prepared according to Affymetrix standard protocol, and hybridized onto the Custom design Vharveyi520694 arrays (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 scanned using the GeneChip 7G Scanner (Affymetrix. GeneChip® Operating Software 1.4 (GCOS 1.4) (Affymetrix) was used to acquire and process array images Microarray Suite 5.0 (MAS5) algorithm, and generated the Report files summarizing
|
| Description |
Gene expression data from V. campbellii at Mid-log phase
|
| Data processing |
CEL files were processed using the GeneChip® Operating Software 1.4 (GCOS 1.4) (Affymetrix) to get signal intensity.
|
| |
|
| Submission date |
Mar 19, 2010 |
| Last update date |
Mar 19, 2010 |
| 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) |
| GSE20960 |
Expression profiling data from mid-log phase cultures of wild type Vibrio campbellii BAA-1116 |
|
