|
| Status |
Public on Aug 22, 2011 |
| Title |
H2-producing_Rhodobacter_lactate_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
H2-producing R. sphaeroides provided lactate & glutamate
|
| Organism |
Cereibacter sphaeroides |
| Characteristics |
organic substrate: lactate
nitrogen source: glutamate
cell density, in klett units (1 ku ~10^7 cells/ml): 320
growth stage: early post-exponential
|
| Treatment protocol |
Cell culture was transferred to an ice-cold conical tube containing 1/8 volume of 5% water-saturated phenol in ethanol. Cells were collected by centrifugation (5,000 X g for 5 min at 4 C); cell pellets were frozen in dry ice/ethanol and stored at -80 C.
|
| Growth protocol |
R. sphaeroides cultures were grown photoheterotrophically in Sistrom's Minimal Medium (with succinate replaced by the indicated organic substrate, and NH4+ replaced by glutamate where indicated) at a temperature of 28-30 C and a light intensity of 10 W/m2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed by addition of 1/10 v 10% SDS at 64 C for 2 min. 1/10 v 1 M Na acetate (pH 5.2), then equal volume of H2O-saturated phenol, were added. Samples were incubated at 64 C for 6 min, with mixing every 30 s, then chilled on ice before centrifugation (16,000 X g for 10 min at 4 C). The aqueous phase was removed and extracted with equal volumes of phenol, then chloroform, then precipitated (1/10 v 3 M Na acetate, 1 mM EDTA, 2 volumes cold ethanol).
|
| Label |
biotin
|
| Label protocol |
Fragmented cDNA was end-labeled with biotin-16-ddUTP using Terminal Transferase for 2 hr at 37 C.
|
| |
|
| Hybridization protocol |
Labeled cDNA samples were hybridized to Rhodobacter sphaeroides GeneChip CustomExpress microarrays (Affymetrix) following product instructions, for 16 h at 45 C on a rotisserie spindle rotating at 60 rpm.
|
| Scan protocol |
Microarrays were washed, stained, and scanned using the Pseudomonas aeruginosa midi-array hybridization with amplification protocols.
|
| Data processing |
Microarray data sets were normalized by Robust Multichip Average (RMA) with background adjustment and quantile normalization. Statistical analysis of normalized data to identify differentially expressed genes used the limma package. Correction for multiple testing was done using Benjamini-Hochberg correction. Differentially expressed genes were defined as those having an adjusted p-value ≤ 0.01 and a fold-change ≥ 2 with respect to those in the reference data sets. All analyses were conducted in the R statistical programming environment (http://www.R-project.org).
|
| |
|
| Submission date |
Jun 08, 2011 |
| Last update date |
Aug 22, 2011 |
| Contact name |
Timothy Donohue |
| E-mail(s) |
tdonohue@bact.wisc.edu
|
| Phone |
608-262-4663
|
| Fax |
608-890-2270
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Department of Bacteriology
|
| Street address |
1550 Linden Drive
|
| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL162 |
| Series (1) |
| GSE29811 |
Expression data from H2-producing Rhodobacter sphaeroides cultures fed different organic substrates |
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