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Status |
Public on Jun 22, 2018 |
Title |
D2095, biological rep1 |
Sample type |
RNA |
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Source name |
D2095 isolate with 17h growth in SM medium
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Organism |
Burkholderia multivorans |
Characteristics |
isolate: D2095 (BM11L)
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Treatment protocol |
Bacterial cells were resuspended in RNAprotect bacteria reagent (Qiagen).
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Growth protocol |
D2095, D2095R, D2214G and D2214P bacterial isolates were grown in 100 ml SM medium contained into 250 ml Erlenmyer flasks at 37ºC, 250 r.p.m. for 17 h
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extraction was carried out using the RNeasy MiniKit (Qiagen) by following manufacturer’s recommendation
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Label |
biotin
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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.
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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.
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Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
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Description |
Mucoid isolate
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Data processing |
Scanned arrays were analyzed with Affymetrix Expression Console software. Subsequent analysis was carried out with DNA-Chip Analyzer 2008. The 12 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 06, 2018 |
Last update date |
Jun 23, 2018 |
Contact name |
Leonilde Morais Moreira |
E-mail(s) |
lmoreira@ist.utl.pt
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Phone |
+351 218419031
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Organization name |
Instituto Superior Tecnico
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Department |
Bioengineering
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Lab |
Biological Sciences
|
Street address |
A. Rovisco Pais
|
City |
Lisboa |
ZIP/Postal code |
1049-001 |
Country |
Portugal |
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Platform ID |
GPL13356 |
Series (1) |
GSE111467 |
Expression data from Burkholderia multivorans mucoid and nonmucoid isolates |
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