|
| Status |
Public on Jan 01, 2017 |
| Title |
E. coli ATCC 25922 Ser83Leu+QnrS replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Exponential cells (DO600=0,4)
|
| Organism |
Escherichia coli ATCC 25922 |
| Characteristics |
substrain: EC24
genotype/variation: Ser83Leu substitution in GyrA and coding for QnrS1
|
| Treatment protocol |
Biological replicates per genotype were incubated at 1 mg/L of ciprofloxacin during 60 minutes (that means 250xMIC for E. coli ATCC 25922, 8xMIC for EC14, 2xMIC for EC19 and 1xMIC for EC24).
|
| Growth protocol |
E. coli ATCC 25922 (wild-type) and EC14, EC19, EC24 (LLQR) isogenic strains were tested to evaluate the global response to relevant fix concentration of ciprofloxacin (1 mg/L, breakpoint for reduced susceptibility according to CLSI). All of them were susceptible to quinolones according to CLSI breakpoints. (CLSI, n.d.) Cultures were started from single colonies and grown overnight in 25 ml of LB. These cell were diluted 1:100 and growth to cell concentration of 4x108 cells/ml (OD600 nm=0.4, exponential phase) for treatment. Three biological replicates per genotype were incubated at 1 mg/L of ciprofloxacin during 60 minutes (that means 250xMIC for E. coli ATCC 25922, 8xMIC for EC14, 2xMIC for EC19 and 1xMIC for EC24). Approximately 109 cells (2 ml) were taken for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, 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.
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| |
|
| 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 Whole Human Genome Oligo Microarrays (G4112A) 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 |
Scanned on an Agilent G2505C scanner. Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
Biological replicate 2 of 2. 1 hour of exposition to 1 mg/L of ciprofloxacin
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
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| |
|
| Submission date |
Sep 01, 2016 |
| Last update date |
Jan 01, 2017 |
| Contact name |
Jose Manuel Rodriguez Martinez |
| E-mail(s) |
jmrodriguez@us.es
|
| Phone |
+34 954552863
|
| Organization name |
University of Seville
|
| Street address |
Snchez Pizjuan SN
|
| City |
Seville |
| ZIP/Postal code |
41009 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13360 |
| Series (1) |
| GSE86341 |
LLQR E. coli ATCC 25922 vs wild-type E. coli ATCC 25922 |
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