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Sample GSM2300527 Query DataSets for GSM2300527
Status Public on Jan 01, 2017
Title E. coli ATCC 25922 QnrS replicate 1
Sample type RNA
 
Source name Exponential cells (DO600=0,4)
Organism Escherichia coli ATCC 25922
Characteristics substrain: EC14
genotype/variation: 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.
 
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 1 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.
 
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

Data table header descriptions
ID_REF
VALUE processed Cy3 signal intensity (Agilent gProcessedSignal)

Data table
ID_REF VALUE
1 1.087885e+002
2 7.410460e+000
3 7.726576e+000
4 8.011908e+000
5 1.380841e+002
6 8.518184e+000
7 8.728160e+000
8 2.411345e+002
9 3.567138e+003
10 3.226427e+002
11 1.011359e+002
12 3.958934e+001
13 9.451815e+000
14 5.341642e+002
15 4.677087e+002
16 2.319633e+001
17 1.986553e+001
18 2.174336e+004
19 3.322808e+001
20 8.042299e+001

Total number of rows: 15744

Table truncated, full table size 296 Kbytes.




Supplementary file Size Download File type/resource
GSM2300527_ATCC_pBKc_Sc1.txt.gz 2.5 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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