|
| Status |
Public on Aug 03, 2012 |
| Title |
RpoHI_FLAG replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Gene expression levels in R. sphaeroides expressing RpoHI_FLAG
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
strain: RSP_2410 mutant with plasmid containing RpoHI_FLAG
growth phase: mid-eponential
grow conditions: aerobic in minimal medium
|
| 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 |
Cells were cultured in minimal succinate based medium under aerobic conditions at 30C until early-exponential growth phase when 100 uM IPTG was added to induce the expression of RpoHI_FLAG for 3 hours before harvesting cells.
|
| 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 |
Jul 29, 2012 |
| Last update date |
Aug 03, 2012 |
| Contact name |
Yann S Dufour |
| Organization name |
University of Wisconsin - Madison
|
| Department |
Bacteriology
|
| Lab |
Timothy Donohue
|
| Street address |
1550 Linden Drive
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL162 |
| Series (2) |
| GSE39712 |
RpoHI and RpoHII regulons in Rhodobacter sphaeroides 2.4.1 from gene expression profiling |
| GSE39806 |
RpoHI and RpoHII regulons in Rhodobacter sphaeroides 2.4.1 |
|
