|
| Status |
Public on Sep 17, 2014 |
| Title |
Amikacin (AMK) resistant strain_Line4 |
| Sample type |
RNA |
| |
|
| Source name |
Exponential culture of AMK resistant strain Line4
|
| Organism |
Escherichia coli |
| Characteristics |
strain: Amikacin (AMK) resistant strain Line4
|
| Treatment protocol |
One hundred eighty uL of exponential cultures were withdrawn rapidly, and cells were killed immediately by the addition of an equal volume of ice-cold ethanol that contained 10% (w/v) phenol. The cells were collected by centrifugation at 20,000 × g at 4 °C for 5 min, and the pelleted cells were stored at −80 °C prior to RNA extraction.
|
| Growth protocol |
Precultures were prepared by shaking -80 °C glycerol stocked parent and evolved resistant strains in 200 uL of modified M9 medium in 96-well microplates for 23 h at 34 °C without antibiotic. The cells precultured were diluted to an OD600 nm of 1 × 10^-4 into 200 uL of fresh modified M9 medium in 96-well microplates. Then, cultures were grown with shaking at 34 °C to an OD600 in the 0.072 ~ 0.135 range (equivalent of 10 generations).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified from cells using an RNeasy micro kit with on-column DNA digestion (Qiagen) in accordance with the manufacturer’s instructions. The quality of the purified RNA was evaluated using Agilent 2100 Bioanalyzer with an RNA 6000 Nano kit (Agilent Technologies). Only purified RNAs that had RIN (RNA integrity number) of 9.0 or more were utilized. The purified RNAs were stored at −80°C prior to transcriptome analysis.
|
| Label |
Cy3
|
| Label protocol |
One hundred ng of purified total RNAs was labeled using the Low Input Quick Amp WT Labeling Kit (Agilent Technologies) with Cyanine3 (Cy3) in accordance with the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
After confirmation of yields (> 825 ng) and specific activities (> 15 pmol/μg) of the Cy3-labbeled cRNAs using NanoDrop ND-2000, 600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x GE Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to custom designed the Agilent 8× 60K arrays for E. coli W3110, E.coli_K12_Expression_Ver.1, for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G4900DA) using one color scan setting for 8x60k array slides (AgilentG3_GX_1Color, Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
AMK4
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: ExternalFullGEML_043017_D_F_20120907) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jul 14, 2014 |
| Last update date |
Sep 17, 2014 |
| Contact name |
Chikara Furusawa |
| E-mail(s) |
chikara.furusawa@riken.jp
|
| Organization name |
RIKEN
|
| Department |
Quantitative Biology Center (QBiC)
|
| Lab |
Laboratory for Multiscale Biosystem Dynamics
|
| Street address |
6-2-3 Furuedai
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0874 |
| Country |
Japan |
| |
|
| Platform ID |
GPL18948 |
| Series (1) |
| GSE59408 |
Prediction of antibiotic resistance by large-scale phenotypic and genotypic data |
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