|
| Status |
Public on Sep 03, 2015 |
| Title |
KT2440(pCAR1Dpmr)_L |
| Sample type |
RNA |
| |
|
| Source name |
cDNA prepared from succinate culture of KT2440(pCAR1Dpmr) in log phase
|
| Organism |
Pseudomonas putida |
| Characteristics |
treatment: cDNA prepared from succinate culture of KT2440(pCAR1Dpmr) in log phase
strain: KT2440
|
| Treatment protocol |
[ChIP-chip analysis] Cells were treated with formaldehyde to a final concentration of 1% to cross-link proteins and DNA.
|
| Growth protocol |
An overnight culture of each strain in LB at 30°C was inoculated into 100 ml NMM-4 supplemented with 0.1% (wt/vol) succinate to obtain an initial turbidity at 600 nm of 0.05 and then incubated at 30°C in a rotating shaker at 120 rpm for 4 h (log phase, a turbidity at 600 nm of 0.15 to 0.25) or 8 h (early-stationary phase, a turbidity at 600 nm of 0.35 to 0.4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
[Transcriptome analysis] Cells were treated with RNA Protect Bacteria reagent (Qiagen, CA, USA). Total RNA was extracted using NucleoSpin RNA II (Macherey-Nagel GmbH & Co. KG, 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, USA) to prevent generation of spurious second-strand cDNA. [ChIP-chip analysis] Cells were disrupted by sonication and His-tagged proteins were purified by QuickPick IMAC metal affinity kit (Bio-Nobile, Turku, Finland). Cross-links were dissociated by heating and the resulting DNA was purified using Qiaquick kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Labelling of the cDNA or DNA was achieved using a GeneChip WT Double-Stranded DNATerminal Labeling Kit (Affymetrix, Santa Clara, CA).
|
| |
|
| Hybridization protocol |
The labelled samples were hybridized individually with each chip using a GeneChip Hybridization Oven 640 (Affymetrix) at 60 rpm and 50°C for 16 h. The chips were then washed and stained using a Hybridization, Wash and Stain Kit (Affymetrix) according to a modified version of the FleX450-0005 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 thePseudomonas putida KT2440 chromosome (RefSeq: NC_002947) at 11-bp density. BPMAP files are provided as supplementary files. KT2440b520511FR-3.bpmap represents the forward strand of KT2440 chromosome, and KT2440b520511FF-3.bpmap represents the reverse strand of KT2440 chromosome.
|
| 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 KT2440b520511FR-3.bpmap for the forward strand and KT2440b520511FF-3.bpmap for the reverse strand; the BAR files with F indicate the forward strand and with R indicate the reverse strand of KT2440 chromosome. Bandwidth: 30, Threshold: 0, MaxGap: 30, MinRun, 30.
KT2440b520511FR-3.bpmap BPMAP for the forward strand of KT2440 chromosome
KT2440b520511FF-3.bpmap BPMAP for the reverse strand of KT2440 chromosome
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| |
|
| Submission date |
Sep 02, 2015 |
| Last update date |
Sep 09, 2015 |
| 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 |
GPL8296 |
| Series (2) |
| GSE72637 |
MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (KT2440) |
| GSE72639 |
MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently |
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