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| Status |
Public on Dec 25, 2012 |
| Title |
KT2440(pCAR1)_6hr_IncP-7 array |
| Sample type |
RNA |
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| Source name |
cDNA prepared from succinate culture of KT2440(pCAR1)
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| Organism |
Pseudomonas putida |
| Characteristics |
strain: KT2440
genotype/variation: pCAR1
growth time: 6hr
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| Growth protocol |
Each strain was grown in 5 ml of LB at 300 strokes/min for 14-14.5 h (pre-culture) and then washed and transferred into 100-ml of NMM-4 buffer (Miyakoshi et al. Transcriptome analysis of Pseudomonas putida KT2440 harboring the completely sequenced IncP-7 plasmid pCAR1. J Bacteriol 2007 Oct;189(19):6849-60. PMID: 17675379) supplemented with 0.1% succinate adjusting its turbidity (600 nm) at 0.05. The resultant culture was incubated on a rotary shaker at 120 rpm and cell growth was monitored in by measuring its turbidity (600 nm).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were treated with RNA Protect Bacteria reagent (Qiagen, CA, USA) to stabilize RNA of them.Total RNA was extracted using NucleoSpin RNA II (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The eluted RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI) at 37˚C for 30 min. After inactivation of DNase by the addition of the provided stop reagent and subsequent incubation at 65˚C for 10 min, total RNA was repurified using NucleoSpin RNA Clean-up (Macherey-Nagel).
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| Label |
biotin
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| Label protocol |
Single-strand cDNA was synthesized in 60 µl of 1× First Strand Buffer (Invitrogen, Carlsbad, CA) containing 12 µg of total RNA, 360ng of actinomycin D (A1410, SIGMA, St.Louis, USA), 750 ng of random primers (Invitrogen), 1500 units of SuperScript II (Invitrogen), 60 units of RNaseOUT (Invitrogen), 10 mM DTT (Invitrogen), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.4 mM dTTP, and 0.1 mM dUTP (Roche Applied Science, Mannheim, Germany). After the RNA and random primers were denatured at 70˚C for 10 min and annealed at 25˚C for 10 min, the remaining reagents were added, and the reaction mixture was then incubated at 25˚C for 10 min, 37˚C for 60 min, 42˚C for 60 min, and 70˚C for 10 min. After cDNA synthesis, template RNA was degraded with one-third volume of 1N NaOH at 65˚C for 30 min; one-third volume of 1N HCl was added to neutralize the reaction mixture before cleanup. The cDNA was purified using a QIAquick PCR Purification Kit (Qiagen). The cDNA was fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix). The purified single-stranded cDNA (~5 µg) was fragmented in 48 µl of 1× cDNA Fragmentation Buffer containing 15 units of Uracil DNA Glycosylase (UDG) and 225 units of Apurinic/apyrimidinic Endonuclease 1 (APE1) at 37˚C for 60 min, and incubated at 93˚C for 2 min. The fragmented cDNA was labeled in 60 µl of 1× TdT Buffer containing 60 units of Terminal Deoxynucleotidyl Transferase (TdT) and 0.083 mM GeneChip DNA Labeling Reagent at 37˚C for 60 min, and incubated at 70˚C for 2 min.
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| Hybridization protocol |
Labeled cDNA was mixed with 50 pM control oligonucleotide B2 (Affymetrix), 1× Hybridization Mix (Affymetrix), and 7% DMSO in a total volume of 200 µl and denatured at 95˚C for 5 min. Per array, 130 µl of the hybridization cocktail was hybridized at 45˚C for 16 h with a rotation rate of 60 rpm using a GeneChip Hybridization Oven 640 (Affymetrix). Chips were washed and stained using a Hybridization, Wash, and Stain Kit (Affymetrix) according to the FlexFS450-0002 protocol of GeneChip Fluidics station 450 (Affymetrix).
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| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
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| Description |
This is an Affymetrix custom-commercial tiling array that covers the IncP-7 carbazole-degradative plasmid pCAR1 (RefSeq: NC_004444.1) 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. The replications of CEL files were together 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 |
Jul 25, 2012 |
| Last update date |
Dec 25, 2012 |
| 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) |
| GSE39636 |
Modulation of primary cell function of host Pseudomonas bacteria by conjugative plasmid pCAR1 |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM973508_081201_pCAR1_KTPC_SUC6h_ActD+.CEL.gz |
448.0 Kb |
(ftp)(http) |
CEL |
| GSM973508_F_081201KTPC_pCAR1_SUC_6h_ActD+_signal.bar.gz |
123.0 Kb |
(ftp)(http) |
BAR |
| GSM973508_R_081201KTPC_pCAR1_SUC_6h_ActD+_signal.bar.gz |
114.4 Kb |
(ftp)(http) |
BAR |
| Processed data provided as supplementary file |
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