|
| Status |
Public on Apr 06, 2016 |
| Title |
optA1_gpt_30 (Low Dilution) Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
optA1 gpt strain w/o hypoxanthine 30 min at low dilution protocol
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
|
| Treatment protocol |
At OD630nm = 0.1, the cultures were filtered through 47-mm diameter polycarbonate membrane filter (0.4 µm pore size; Millipore), washed with the same medium without hypoxanthine (Hx), and then resuspended at the same cell density (OD630 = 0.1) in the medium without Hx, and growth was continued as before. The OD630 was followed closely during the next two hours, and the cells were periodically diluted with fresh, pre-warmed medium without Hx to keep OD between values 0.1 and 0.3 (low-dilution protocol). Samples were taken for microarray analysis (see below) at 0, 15, 30, 45, 60 and 120 minutes. In a parallel procedure, the optA1 gpt double mutant was also followed in a near-identical manner using a more highly diluted culture over a six-hour incubation period in the absence of hypoxanthine (high-dilution protocol). The effectiveness of the latter treatment was followed microscopically by observing the extensive cellular filamentation associated with dGTP starvation.
|
| Growth protocol |
The strains were grown exponentially (after initiation by a 1,000-fold dilution from overnight cultures) for ~5 generations at 37°C in Vogel-Bonner (VB) minimal medium supplemented with glucose (0.4%), pantothenic acid (5 µg/ml), casamino acids (Becton-Dickenson) (1%), and hypoxanthine (50 µg/ml).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At each time point, 15 ml of each culture was mixed with 30 ml of RNAprotect Bacteria Reagent (Qiagen), vortexed for 5s, incubated for 5 min at room temperature, and centrifuged for 10 min at 5000g. The pellet was processed using a Qiagen RNeasy Midi kit with on-column DNase digestion using Qiagen DNase. The final elution of RNA from the column was with 160 μl RNase-free water.
|
| Label |
biotin
|
| Label protocol |
Total RNA was amplified as directed in the Affymetrix Prokaryotic Target Labeling Assay protocol.
|
| |
|
| Hybridization protocol |
1 μg of amplified biotin-aRNAs were fragmented and hybridized to each array for 16 hours at 45°C in a rotating hybridization oven using the Affymetrix Target Hybridization Controls and protocol. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit and user manual.
|
| Scan protocol |
Arrays were scanned in an Affymetrix Scanner 3000.
|
| Description |
Gene expression from optA1 gpt strain w/o hypoxanthine 30 min at low dilution protocol
|
| Data processing |
Data was obtained using the GeneChip® Command Console and Expression Console Software (AGCC; Version 3.2 and Expression Console; Version 1.2) using the MAS5 algorithm to generate .CHP files.
|
| |
|
| Submission date |
Apr 06, 2016 |
| Last update date |
Apr 06, 2016 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
microarray@niehs.nih.gov, liuliw@niehs.nih.gov
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE80002 |
Transcriptome analysis of Escherichia coli during dGTP Starvation |
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