|
| Status |
Public on Dec 01, 2012 |
| Title |
IST408, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
IST408 wild-type with 12h growth in S medium
|
| Organism |
Burkholderia cepacia |
| Characteristics |
isolate: IST408
genotype: wild-type
|
| Treatment protocol |
Bacterial cells were resuspended in RNAprotect Bacteria Reagent (Qiagen).
|
| Growth protocol |
Bacterial strains were grown in 100 ml S medium in 250 ml Erlenmeyer flasks at 30ºC, 250 r.p.m. for 12 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was carried out using the RNeasy MiniKit (Qiagen) by following the manufacturer's recommendations.
|
| 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 |
B. cepacia_IST408_2
Mucoid wild-type clinical strain.
|
| Data processing |
DNA-based probe-selection was performed using Xspecies DNA hyb CDF batchmaker v3.2, available here: http://affymetrix.arabidopsis.info/xspecies/. An optimal signal cutoff of 80 and minimum number of probes per probe set of 7 were determined empirically, resulting in the masking of 24223 of the original 226576 probes on the array and the exclusion of 156 probe sets. The new cdf file created (Bcc1sa520656F_Xspecies.CEL80.cdf) was used in DNA-Chip Analyzer 2008 for the subsequent gene expression analyses.
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 (LCB) between experiment and baseline was above 1.2 when the bceF::Tp mutant was compared with the wild-type IST408 and 1.6 for the comparison between the bceE::Tp mutant and IST408.
|
| |
|
| Submission date |
May 23, 2012 |
| Last update date |
Dec 01, 2012 |
| 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 |
GPL15600 |
| Series (1) |
| GSE38183 |
Expression data from Burkholderia cepacia IST408 bceE and bceF mutants |
|
