|
| Status |
Public on May 10, 2016 |
| Title |
Mutant B8_0.8%Bu_1.5h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mutant B8, 0.8%butanol,1.5h,replicate 2
|
| Organism |
Escherichia coli |
| Characteristics |
genotype/variation: σ70 mutation
phenotype: 2% n-butanol tolerance
|
| Treatment protocol |
n-Butanol (0.8%, v/v) was added at 0.8 OD660 and further incubated for 1.5 h.
|
| Growth protocol |
E. coli strains harboring σ70 mutant B8 and WT were cultured overnight and inoculated (1%) into fresh medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen RNeasy kit (Hilden, Germany) following manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (QIAGEN, GmBH, Germany) and RNase‐Free DNase Set (QIAGEN, GmBH, Germany).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-020097 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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 1.5hr under 0.8% (v/v) butanol,
|
| Data processing |
Data was extracted with Agilent Feature Extraction software 10.7 (Agilent) using default parameters (protocol GE1-v1_91) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Mar 16, 2016 |
| Last update date |
May 10, 2016 |
| Contact name |
Ye Ni |
| E-mail(s) |
yni@jiangnan.edu.cn
|
| Organization name |
Jiangnan University
|
| Department |
School of Biotechnology
|
| Street address |
1800 Lihu Road
|
| City |
Wuxi |
| State/province |
Jiangsu |
| ZIP/Postal code |
214122 |
| Country |
China |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE79305 |
DNA Microarray of Global Transcription Factor Mutant Reveals Membrane-Related Proteins Involved in n-Butanol Tolerance in Escherichia coli |
|
