|
| Status |
Public on Mar 30, 2018 |
| Title |
PaBt-Lact-C_rep_2 |
| Sample type |
RNA |
| |
|
| Source name |
Pa & Bt cultured in MOPS lactate for 4 h (control condition), biological replicate 2
|
| Organisms |
Burkholderia thailandensis E264; Pseudomonas aeruginosa PA14 |
| Characteristics |
condition: MOPS Lactate
replicate: C
|
| Treatment protocol |
Following completion of the gene induction period, the cells from each culture were gathered via centrifiguation, resuspended in 800µL of ~70˚C RNAzol, and frozen at -80˚C until extraction.
|
| Growth protocol |
Strains were first grown overnight in MOPS minimal supplemented with lactate as a carbon source. The following day, cells from these cultures were adjusted to the same optical density, and then cultured for 4 h in both MOPS lactate media and MOPS lactate media supplemented with purified pulmonary surfactant (Survanta).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was first extracted using RNAzol and Zymo's RNA mini prep kit. Small RNAs were then removed through a second extraction via Qiagen's RNEasy kit & provided protocol. RNA quality was assessed via Agilent BioAnalyzer and quantified through Qubit fluorometer.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA was prepared from 50 ng of total RNA using the NuGen Ovation Pico system and the Encore Biotin Module according to manufacturer's instructions. For each cDNA sample prepared, the input consisted of 1:1 mixtures of either P.aeruginosa and B. thailandensis, or K. pneumoniae and S. maltophilia total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 2.4 ug of cRNA were hybridized on the Custom KpSmPaBc Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix 7G Scanner.
|
| Data processing |
The data were processed via RMA using Affymetrix's Expression Console and default settings. Statistical analysis and further data assessment was then performed using Affymetrix's transcriptome analysis console version 3.0. Genes exhibiting at least a 2.5 fold change in transcript abundance between conditions with an ANOVA p value <0.05 were considered significantly altered by surfactant. To obtain the data reflected in the associated manuscpript, the affymetrix gene cluster ID column was filtered to obtain the klebsiella-specific probe intensities by restricting the displayed IDs to those beginning with the Kp MGH 78578 genebank chromosome and plasmid accession numbers (CP000647,CP000648, CP000649, CP000650, CP000651, and CP000652) . Probe values specific to non-coding regions of the genome were also removed by filtering the description column to those not containing the word intergenic region. This methodology can also be used to obtain Pseudomonas aeruginosa UCBS PA14 (CP000468), Burkholderia thailandensis E264 (CP000845 & CP000846), and Stenotrophomonas maltophilia K279A (AM743169)-specific probe data.
|
| |
|
| Submission date |
Feb 14, 2018 |
| Last update date |
Mar 30, 2018 |
| Contact name |
Matthew J Wargo |
| E-mail(s) |
matthew.wargo@med.uvm.edu
|
| Organization name |
University of Vermont
|
| Department |
Department of Microbiology and Molecular Genetics
|
| Street address |
95 Carrigan Dr
|
| City |
Burlington |
| State/province |
VT |
| ZIP/Postal code |
05405 |
| Country |
USA |
| |
|
| Platform ID |
GPL24615 |
| Series (1) |
| GSE110628 |
Transcriptional responses of K. pneumoniae, S. maltophilia, P.aeruginosa, and B. thailandensis exposed to lung surfactant |
|
