|
| Status |
Public on Oct 17, 2017 |
| Title |
P-Co |
| Sample type |
RNA |
| |
|
| Source name |
Parent with Co stress
|
| Organism |
Escherichia coli str. K-12 substr. MDS42 |
| Characteristics |
strain: Parent
stress: Co
|
| Treatment protocol |
Exponential cultures were withdrawn rapidly, and cells were killed immediately by the addition of an equal volume of ice-cold ethanol that contained 10% (w/v) phenol.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 |
The cells were precultured for 60 hours under the same condition with the laboratory with or without the corresponding stressors. Then, 5×10e7 cells in the exponential growth phase were used to transcriptome analysis.
|
| 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 |
Transcriptome data of Parent with Co stress
|
| 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.
Log10 normalized expression of ORF. ORF level expression was calculated by median value of all probes corresponding to each ORF.
|
| |
|
| Submission date |
Nov 10, 2016 |
| Last update date |
Oct 17, 2017 |
| Contact name |
Chiara Furusawa |
| E-mail(s) |
chikara.furusawa@riken.jp
|
| Phone |
+81-(0)6-6155-0456
|
| Organization name |
RIKEN Quantitative Biology Center
|
| Street address |
Furuedai
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0874 |
| Country |
Japan |
| |
|
| Platform ID |
GPL18948 |
| Series (1) |
| GSE89746 |
Prediction of Cross-resistance and Collateral Sensitivity by Gene Expression profiles and Genomic Mutations. |
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