|
| Status |
Public on Jan 01, 2014 |
| Title |
J2315_hfq2, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
J2315 Δhfq2 with 16h growth in LB medium
|
| Organism |
Burkholderia cenocepacia |
| Characteristics |
isolate: J2315 Δhfq2
|
| Treatment protocol |
Bacterial cells were resuspended in RNAlater reagent (Ambion).
|
| Growth protocol |
Bacterial strains were grown in 150 ml L medium contained into 250 ml Erlenmyer flasks at 37ºC, 250 r.p.m. for 16 h
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was carried out using the using the miRVana miRNA extraction kit (Ambion), and total RNA was enriched with the small-sized RNAs fraction, following manufacturer’s recommendation
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix custom dual species Burkholderia arrays, according to the manufacturer’s Prokaryotic Target Preparation Assay. Briefly, 10 ug of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix, Santa Clara, CA) was used in a reverse transcription reaction with random primers (Invitrogen Life Technologies) to generate first-strand cDNA. After removal of RNA, 2 ug of cDNA were fragmented with DNase and end-labeled with biotin using terminal polynucleotidyl transferase (GeneChip WT Terminal Labeling Kit; Affymetrix). Size distribution of the fragmented and end-labeled cDNA, was assessed using an Agilent 2100 Bioanalyzer.
|
| |
|
| Hybridization protocol |
Following fragmentation, 2 µg of end-labeled fragmented cDNA were used in a 200-µl hybridization cocktail containing added hybridization controls and hybridized on arrays for 16 hours at 50ºC. Modified post-hybridization wash and double-stain protocols (FLEX450_0005; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Mutation in the hfq2 gene
|
| Data processing |
Scanned arrays were analyzed with Affymetrix Expression Console software for quality control. Subsequent analysis was carried out with DNA-Chip Analyzer 2010. First a digital mask was applied, leaving for analysis only the 8812 probe sets on the array representing Burkholderia cenocepacia J2315 transcripts. Then the 9 arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the arrays were used to obtain model-based gene expression indices based on a Perfect Match (PM)-only model. Replicate data (triplicates) for each bacterial isolate was weighted gene-wise by using inverse squared standard error as weights. All genes compared were considered to be differentially expressed if the 90% lower confidence bound of the fold change between experiment and baseline was above 1.2.
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| |
|
| Submission date |
May 23, 2013 |
| Last update date |
Jan 01, 2014 |
| Contact name |
Leonilde Morais Moreira |
| E-mail(s) |
lmoreira@ist.utl.pt
|
| Phone |
+351 218419031
|
| Organization name |
Instituto Superior Tecnico
|
| Department |
Bioengineering
|
| Lab |
Biological Sciences
|
| Street address |
A. Rovisco Pais
|
| City |
Lisboa |
| ZIP/Postal code |
1049-001 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL13356 |
| Series (1) |
| GSE47344 |
Expression data from Burkholderia cenocepacia J2315 hfq and hfq2 mutants |
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