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Sample GSM2572227 Query DataSets for GSM2572227
Status Public on Apr 11, 2017
Title PF-KT_pCAR1_with_cation_2
Sample type RNA
 
Source name cDNA prepared from Pf0-1(pCAR1) and KT2440 incubated in CF buffer with cation
Organisms Pseudomonas putida KT2440; Pseudomonas fluorescens Pf0-1
Characteristics strain: Pf0-1
strain: KT2440
Treatment protocol The cells were suspended in 4 mL CF buffer and the turbidity of donor and the recipient was adjusted to 2.0 at 600 nm. As for the mixture samples of donor and recipient, each 4 mL suspension was mixed in the 50 mL tube. For the donor and recipient samples, the 4 mL of each culture was mixed with another 4 mL CF buffer in 50 mL tube. The concentration of the cations was set to 0 μM or 400 μM (CaCl2 and MgSO4), respectively. The mating duration was 24 h.
Growth protocol Donor (Pseudomonas fluorescens Pf0-1) and recipient (Pseudomonas putida KT2440) were grown overnight in 1/2LB. The cells were harvested by centrifugation and washed five times by CF buffer.
Extracted molecule total RNA
Extraction protocol The cells were treated with RNA Protect Bacteria reagent (Qiagen, CA, USA). Total RNA was extracted using NucleoSpin RNA II (Macherey-Nagel, 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, 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 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) 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.
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 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.
 
Submission date Apr 10, 2017
Last update date Apr 11, 2017
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 (1)
GSE97565 Divalent cations promote the transfer of the IncP-7 plasmid pCAR1 among different Pseudomonas hosts

Supplementary file Size Download File type/resource
GSM2572227_20140610_FLACvsKT_pCAR1_with_cation2.CEL.gz 404.1 Kb (ftp)(http) CEL
GSM2572227_20140610_FLACvsKT_pCAR1_with_cation2_F_signal.bar.gz 111.7 Kb (ftp)(http) BAR
GSM2572227_20140610_FLACvsKT_pCAR1_with_cation2_R_signal.bar.gz 107.4 Kb (ftp)(http) BAR
Processed data provided as supplementary file

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