|
| Status |
Public on Nov 06, 2022 |
| Title |
Untreated control culture, biological replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
Planktonic PAO1∆rpoH (PBAD-rpoH) cells, replicate 1
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
induction of rpoh: Non-induced
|
| Treatment protocol |
For rpoH induction in P. aeruginosa PAO1∆rpoH (PBAD-rpoH), following 7.75 h of growth in BMM, arabinose was added to a final concentration of 0.1%. After 15 minutes, cells were collected from the non-treated control and arabinose treated cultures by centrifugation at 9k RPM for 2 min at 4C. The cell pellets were frozen at -80C for RNA extraction.
|
| Growth protocol |
The PAO1(PBAD-rpoH) strain was cultivated overnight at 37C in Tryptic Soy Broth amended with 40 µg x ml-1 tetracycline and 0.1% arabinose. Two 1 ml aliquots of the overnight culture were washed twice with 1 ml phosphate buffered saline (PBS) pH 7.0 (9k RPM, 1 min) and resuspended in 1 ml PBS. Each washed aliquot of the overnight culture was added to 25 ml of Biofilm Minimal Medium (BMM) in a 125 ml baffled flask and incubated with shaking at 200 rpm at 37C for 7.75 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from cells using the hot phenol extraction method with chemicals obtained from Fisher Scientific (Waltham, MA). Briefly, pelleted cells were resuspended in 200 µl lysis buffer (0.15 M sucrose, 0.01 M sodium acetate, pH 4.5) and 200 µl 2% sodium dodecyl sulfate. Following the addition of 400 µl phenol, the mixture was incubated for 5 min at 65C with frequent vortexing. A Heavy Phase Lock Gel (PLG) tube (5 Prime Therapeutics, South San Francisco, CA) was used according to the manufacturer’s instructions to separate the phases. A second cleanup was performed in the PLG tube with 400 µl phenol:chloroform:isoamyl alcohol (25:24:1) solution. The aqueous phase was removed, and the RNA was purified using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) with an on-column DNase treatment according to the manufacturer’s instructions. The purified RNA was then treated with turbo-DNase using the Turbo-DNA free kit (Invitrogen, now Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. RNA quality was assessed on the Bioanalyzer 2100 using an RNA6000 nano assay (Agilent Technologies, Santa Clara, CA), and all samples were found to be intact with RNA Integrity Number (RIN) scores > 9.
|
| Label |
Biotin
|
| Label protocol |
For each sample, 8 μg of Turbo DNase-treated RNA was reverse transcribed, fragmented, and biotin end labeled according to the Affymetrix prokaryotic target labeling protocol (GeneChip expression analysis technical manual, November 2004).
|
| |
|
| Hybridization protocol |
Labeled cDNA was hybridized to Affymetrix P. aeruginosa microarrays (part 900339) for 16 h at 50C with constant rotation. Microarrays were stained using a GCOS Fluidics Station 450.
|
| Scan protocol |
Microarrays were scanned with an Affymetrix 7G scanner.
|
| Description |
Gene expression data from a noninduced control sample
|
| Data processing |
Affymetrix GCOS v1.4 was used to generate CEL files, which were imported into FlexArray v1.6.1 for quality control, normalization, and data analysis. Microarray data from three biological replicates of 8-hour planktonic arabinose induced PAO1∆rpoH (PBAD-rpoH) cultures and three non-induced PAO1∆rpoH (PBAD-rpoH) cultures were background corrected and normalized using the GC-RMA algorithm in FlexArray v1.6.1. Genes with statistically significant changes in expression (> 2-fold change at p <0.05) were determined by analysis of variance (ANOVA).
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| |
|
| Submission date |
Nov 03, 2022 |
| Last update date |
Nov 07, 2022 |
| Contact name |
Michael J Franklin |
| Organization name |
Montana State University
|
| Department |
Microbiology and Cell Biology
|
| Lab |
Franklin Lab
|
| Street address |
109 Lewis Hall
|
| City |
Bozeman |
| State/province |
MT |
| ZIP/Postal code |
59717 |
| Country |
USA |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE217157 |
Identification of genes regulated by rpoH in Pseudomonas aeruginosa |
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