|
| Status |
Public on Oct 12, 2020 |
| Title |
L-Homoserine (HSE) evolved strain Line4 |
| Sample type |
RNA |
| |
|
| Source name |
HSEE4
|
| Organism |
Escherichia coli |
| Characteristics |
genetic background: MDS42
strain type: Evolved
resistance: HSE resistance
|
| Treatment protocol |
Cells were inoculated from the frozen glycerol stock into 96-well microplates containing 200 µl of modified M9 medium without chemicals used for laboratory evolution and cultivated 24h at 34°C with agitation at 900 rotations/min. The precultures were diluted to an OD620 of 0.000033-0.0004 into 200 µl of fresh modified M9 medium in 96-well microplates and cultured. After 12 to 21 h incubation, the exponentially growing cultures (OD600 in the 0.072-0.135 range) were harvested for total RNA extraction.
|
| Growth protocol |
MDS42 and the evolved strains cells were inoculated from the frozen glycerol stock into 5 ml of modified M9 medium in test tubes and cultivated overnight at 34°C at 150 rpm. The overnight cultures were diluted to an OD600 of 0.1 into fresh modified M9 medium in test tubes and cultured.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An equal volume of ice-cold ethanol containing 10% (w/v) phenol was added directly to the exponentially growing cultures to stabilize cellular RNA. The cells were harvested by centrifugation at 20,000 × g at 4°C for 5 min, and the pelleted cells were stored at –80°C before RNA extraction. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The purified RNA samples were stores at – 80°C before microarray experiments. 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) of9.0 or more were utilized. The purified RNAs were stored at −80°C prior to transcriptome analysis.
|
| Label |
Cy3
|
| Label protocol |
100 ng of purified total RNA samples were labelled using the Low Input Quick Amp WT Labeling Kit (Agilent Technologies) with Cyanine3 (Cy3) according to 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 manufacturer's 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 |
Gene expression of E. coli cells at exponential growth phase.
|
| 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.
ORF level expression was calculated by median value of all probes corresponding to each ORF.
|
| |
|
| Submission date |
Sep 12, 2019 |
| Last update date |
Oct 13, 2020 |
| 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) |
| GSE137348 |
High-throughput laboratory evolution reveals global evolutionary constraints for antibiotic resistance |
|
