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Sample GSM1867047 Query DataSets for GSM1867047
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 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
 
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

Supplementary file Size Download File type/resource
GSM1867047_090227_KT2440dPP3765_pCAR1_chr_4h_01.CEL.gz 11.5 Mb (ftp)(http) CEL
GSM1867047_KT2440d3765_pCAR1_chr_4h_01_F_signal.bar.gz 2.5 Mb (ftp)(http) BAR
GSM1867047_KT2440d3765_pCAR1_chr_4h_01_R_signal.bar.gz 2.5 Mb (ftp)(http) BAR
Processed data provided as supplementary file

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