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Sample GSM3015502 Query DataSets for GSM3015502
Status Public on Feb 17, 2018
Title #4_RP4-harboring_8h_1
Sample type RNA
 
Source name cDNA prepared from KT2440 (substrain #4) harboring RP4 plasmid sampled in stationary phase
Organism Pseudomonas putida
Characteristics strain: KT2440
Treatment protocol Cells were treated with RNA Protect Bacteria reagent (Qiagen, CA, USA) to stabilize RNA of them.
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) until its log phase (4 h after inoculation) and early stationary phase (8 h after inoculation).
Extracted molecule total RNA
Extraction protocol 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). 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).
 
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 the Pseudomonas 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 Feb 16, 2018
Last update date Feb 17, 2018
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 (1)
GSE110733 Iron acquisition system and cell shape of host bacteria commonly respond to the carriage of three plasmids belonging to different incompatibility groups.

Supplementary file Size Download File type/resource
GSM3015502_121108_KT2440_RP4__4-1_SUC_8h.CEL.gz 9.5 Mb (ftp)(http) CEL
GSM3015502_F-121108_KT2440_RP4__4-1_SUC_8h_signal.bar.gz 2.2 Mb (ftp)(http) BAR
GSM3015502_R-121108_KT2440_RP4__4-1_SUC_8h_signal.bar.gz 2.2 Mb (ftp)(http) BAR
Processed data provided as supplementary file

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