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| Status |
Public on Apr 11, 2017 |
| Title |
PF_pCAR1_with_cation_2 |
| Sample type |
RNA |
| |
|
| Source name |
cDNA prepared from Pf0-1(pCAR1) incubated in CF buffer with cation
|
| Organism |
Pseudomonas fluorescens Pf0-1 |
| Characteristics |
strain: Pf0-1
|
| 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.
|
| 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.
|
| 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.
|
| Label |
biotin
|
| 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)
|
| Scan protocol |
Signals were detected using a GeneChip Scanner 3000 7G (Affymetrix).
|
| 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.
|
| 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
|
| Phone |
+81-3-5841-3067
|
| Organization name |
The University of Tokyo
|
| Department |
Biotechnology Research Center
|
| Lab |
Laboratory of Environmental Biochemistry
|
| Street address |
1-1-1 Yayoi, Bunkyo-ku
|
| City |
Tokyo |
| ZIP/Postal code |
113-8657 |
| Country |
Japan |
| |
|
| 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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