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Sample GSM1867056 Query DataSets for GSM1867056
Status Public on Sep 03, 2015
Title KT2440DturB(pCAR1)_L
Sample type RNA
 
Source name cDNA prepared from succinate culture of KT2440DturB(pCAR1) in log phase
Organism Pseudomonas putida
Characteristics treatment: cDNA prepared from succinate culture of KT2440DturB(pCAR1) 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 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_004444.1) at 9-bp density.
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.
pCAR1_8b520435FR-3.bpmap BPMAP for the forward strand of pCAR1
pCAR1_8b520435FF-3.bpmap BPMAP for the reverse strand of pCAR1
 
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 GPL6591
Series (2)
GSE72638 MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (pCAR1)
GSE72639 MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently

Supplementary file Size Download File type/resource
GSM1867056_090226_KT2440dPP3765_pCAR1_pCAR1_4h_01.CEL.gz 483.8 Kb (ftp)(http) CEL
GSM1867056_KT2440dPP3765_pCAR1_pCAR1_4h_01_F_signal.bar.gz 124.8 Kb (ftp)(http) BAR
GSM1867056_KT2440dPP3765_pCAR1_pCAR1_4h_01_R_signal.bar.gz 118.2 Kb (ftp)(http) BAR
Processed data provided as supplementary file

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