|
| Status |
Public on Jul 28, 2017 |
| Title |
ATCC 17616_WT SM, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
ATCC 17616 wild-type grown in SM medium (20 g/l D-mannitol) for 22 hours
|
| Organism |
Burkholderia multivorans |
| Characteristics |
genotype: ATCC 17616
|
| Treatment protocol |
Bacterial cells were resuspended in RNAprotect bacteria reagent (Qiagen).
|
| Growth protocol |
Wild-type ATCC 17616 and delta Bmul_2557 mutant were grown overnight in LB medium and inoculated into 50 ml S medium supplemented with 20 g/l of D-glucose (with OD600nm 0.1) contained into 100 ml Erlenmyer flasks at 37ºC, 200 r.p.m. for 22 h, followed by RNA extraction. The wild-type B. multivorans ATCC 17616 was also inoculated into 50 ml of S medium supplemented with 20 g/l D-mannitol (SM medium) and incubated under the same condiotions prior to RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was carried out using the RNeasy MiniKit (Qiagen) by following manufacturer’s recommendation
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix Burkholderia dual species Genome 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 |
wild-type strain
|
| Data processing |
Scanned arrays were analyzed with Affymetrix Expression Console software. Subsequent analysis was carried out with DNA-Chip Analyzer 2008. First a digital mask was applied, leaving for analysis only the 9291 probe sets on the array representing Burkholderia multivorans ATCC 17616 transcripts. Then the 6 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 |
Mar 24, 2017 |
| Last update date |
Jul 28, 2017 |
| 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) |
| GSE97006 |
Expression data from Burkholderia multivorans ATCC 17616 wild-type and Bmul_2557 (ldhR) gene deletion mutant |
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