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Status |
Public on Apr 11, 2017 |
Title |
PF-KT_pCAR1_with_cation_2 |
Sample type |
RNA |
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Source name |
cDNA prepared from Pf0-1(pCAR1) and KT2440 incubated in CF buffer with cation
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Organisms |
Pseudomonas putida KT2440; Pseudomonas fluorescens Pf0-1 |
Characteristics |
strain: Pf0-1 strain: KT2440
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Treatment protocol |
The cells were suspended in 4 mL CF buffer and the turbidity of donor and the recipient was adjusted to 2.0 at 600 nm. As for the mixture samples of donor and recipient, each 4 mL suspension was mixed in the 50 mL tube. For the donor and recipient samples, the 4 mL of each culture was mixed with another 4 mL CF buffer in 50 mL tube. The concentration of the cations was set to 0 μM or 400 μM (CaCl2 and MgSO4), respectively. The mating duration was 24 h.
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Growth protocol |
Donor (Pseudomonas fluorescens Pf0-1) and recipient (Pseudomonas putida KT2440) were grown overnight in 1/2LB. The cells were harvested by centrifugation and washed five times by CF buffer.
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Extracted molecule |
total RNA |
Extraction protocol |
The cells were treated with RNA Protect Bacteria reagent (Qiagen, CA, USA). Total RNA was extracted using NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) and RNeasy Midi kit (Qiagen). The eluted RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI). After inactivation of DNase by the addition of the provided stop reagent and subsequent incubation, total RNA was repurified using NucleoSpin RNA Clean-up (Macherey-Nagel). cDNA was synthesized using SuperScript II (Invitrogen, Carlsbad, CA) in the presence of actinomycin D (Sigma, St.Louis, MO) to prevent generation of spurious second-strand cDNA.
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Label |
biotin
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Label protocol |
Labelling of the cDNA was achieved using a GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix, Santa Clara, CA).
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Hybridization protocol |
The labelled samples were hybridized individually with each chip using a GeneChip Hybridization Oven 640 (Affymetrix) at 60 rpm and 45°C for 16 h. The chips were then washed and stained using a Hybridization, Wash and Stain Kit (Affymetrix) according to the FlexFS450-0002 protocol of the GeneChip Fluidics station 450 (Affymetrix)
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Scan protocol |
Signals were detected using a GeneChip Scanner 3000 7G (Affymetrix).
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Description |
This is an Affymetrix custom-commercial tiling array that covers the IncP-7 carbazole-degradative plasmid pCAR1 (RefSeq: NC_004444) at 9-bp density. BPMAP files are provided as supplementary files. pCAR1_8b520435FR-3.bpmap represents the forward strand of pCAR1, and pCAR1_8b520435FF-3.bpmap represents the reverse strand of pCAR1.
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Data processing |
The CEL files were generated using Affymetrix GCOS. Data was quantile normalized and analyzed with Affymetrix Tiling Analysis Software v1.1, which uses non-parametric quantile normalization and a Hodges-Lehmann estimator for fold enrichment.The intensities were linearly scaled so that the median was 100. Each CEL file was converted into the BAR files using pCAR1_8b520435FR-3.bpmap for the forward strand and pCAR1_8b520435FF-3.bpmap for the reverse strand; the BAR files with F indicate the forward strand and with R indicate the reverse strand of pCAR1. Bandwidth: 30, Threshold: 0, MaxGap: 30, MinRun, 30.
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Submission date |
Apr 10, 2017 |
Last update date |
Apr 11, 2017 |
Contact name |
Hideaki Nojiri |
E-mail(s) |
anojiri@mail.ecc.u-tokyo.ac.jp
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Phone |
+81-3-5841-3067
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Organization name |
The University of Tokyo
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Department |
Biotechnology Research Center
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Lab |
Laboratory of Environmental Biochemistry
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Street address |
1-1-1 Yayoi, Bunkyo-ku
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City |
Tokyo |
ZIP/Postal code |
113-8657 |
Country |
Japan |
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Platform ID |
GPL6591 |
Series (1) |
GSE97565 |
Divalent cations promote the transfer of the IncP-7 plasmid pCAR1 among different Pseudomonas hosts |
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