|
| Status |
Public on May 25, 2010 |
| Title |
KTPC_pCAR1_Pmr_His_4h |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
His-tag ChIP DNA in P.putida KT2440 (pCAR1); Pmr-His
|
| Organism |
Pseudomonas putida |
| Characteristics |
strain: KT2440
antibody: Anti His-tag
antibody manufacturer: GE Healthcare Bio-Sciences, NJ, USA
antibody catalog number: 27-4710-01
|
| Growth protocol |
An overnight culture of KT2440(pCAR1) derivative expressing 6 × His-tagged Pmr in LB at 30˚C was inoculated into 200 ml of NMM-4 supplemented with 0.1 % (v/w) succinate to obtain an initial turbidity (600 nm) at 0.05, and then incubated at 30˚C in a rotating shaker at 120 rpm for 4 h to turbidity (600 nm) at 0.20-0.30.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The His-tagged Pmr and DNA in the cells were in vivo cross-linked by addition of formaldehyde to a final concentration of 1 % for 15 min while shaking at 30˚C. The cross-linking reaction was quenched by addition of glycine to a final concentration of 125 mM for 5 min, then cells were washed twice with chilled TE buffer (pH 8.0). The resultant harvested cells were disrupted by sonication on ice in 2.4 mL of QuickPick IMAC (Bio-Nobile, Truku, Finland) wash buffer. After centrifugation at 17,000×g for 20 min, the supernatant was affinity-purified using the QuickPick IMAC metal affinity kit to yield 6 × His-tagged Pmr complex according to the manufacturer’s instructions. Cross-links were dissociated by heating at 65˚C for 4 h and the resultant DNA was purified using Qiaquick (QIAGEN) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically fragmented and labeled using the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
Input DNA from P.putida KT2440 (pCAR1); Pmr-His
|
| Organism |
Pseudomonas putida |
| Characteristics |
strain: KT2440
fraction: input
|
| Growth protocol |
An overnight culture of KT2440(pCAR1) derivative expressing 6 × His-tagged Pmr in LB at 30˚C was inoculated into 200 ml of NMM-4 supplemented with 0.1 % (v/w) succinate to obtain an initial turbidity (600 nm) at 0.05, and then incubated at 30˚C in a rotating shaker at 120 rpm for 4 h to turbidity (600 nm) at 0.20-0.30.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The His-tagged Pmr and DNA in the cells were in vivo cross-linked by addition of formaldehyde to a final concentration of 1 % for 15 min while shaking at 30˚C. The cross-linking reaction was quenched by addition of glycine to a final concentration of 125 mM for 5 min, then cells were washed twice with chilled TE buffer (pH 8.0). The resultant harvested cells were disrupted by sonication on ice in 2.4 mL of QuickPick IMAC (Bio-Nobile, Truku, Finland) wash buffer. After centrifugation at 17,000×g for 20 min, the supernatant was affinity-purified using the QuickPick IMAC metal affinity kit to yield 6 × His-tagged Pmr complex according to the manufacturer’s instructions. Cross-links were dissociated by heating at 65˚C for 4 h and the resultant DNA was purified using Qiaquick (QIAGEN) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically fragmented and labeled using the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
Labeled DNA was mixed with 50 pM control oligonucleotide B2 (Affymetrix), 1× Hybridization Mix (Affymetrix), and 7% DMSO in a total volume of 200 µl and denatured at 95˚C for 5 min. Per array, 200 µl of the hybridization cocktail was hybridized at 45˚C for 16 h with a rotation rate of 60 rpm using a GeneChip Hybridization Oven 640 (Affymetrix). Chips were washed and stained using a Hybridization, Wash, and Stain Kit (Affymetrix) according to the the modified FleX450-0005 protocol (for chromosomal tiling array, changed temperature of the hybridization to 50˚C) of GeneChip Fluidics station 450 (Affymetrix).
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
Pmr binding sites detection on the 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. The replications of CEL files were together converted into the BAR files using KT2440b520511FF-3; Bandwidth: 300, Threshold: 0, MaxGap: 30, MinRun, 30.
bar files are generated by Affymetrix Tiling Analysis Software v1.1. File KT2440_p_value20.bed and pCAR1_p_value20.bed contains all Pmr binding sites represented in this paper
|
| |
|
| Submission date |
May 24, 2010 |
| Last update date |
May 25, 2010 |
| Contact name |
Hideaki NOJIRI |
| E-mail(s) |
anojiri@mail.ecc.u-tokyo.ac.jp
|
| Phone |
+81-3-5841-3064
|
| Fax |
+81-3-5841-8030
|
| URL |
http://park.itc.u-tokyo.ac.jp/biotec-res-ctr/kampo/eng/index.html
|
| 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) |
| GSE21968 |
Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function |
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