|
| Status |
Public on Feb 17, 2018 |
| Title |
#2_plasmid-free_8h_2 |
| Sample type |
RNA |
| |
|
| Source name |
cDNA prepared from plasmid-free KT2440 (substrain #2) 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. |
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