Comment[ArrayExpressAccession] E-GEOD-57084 MAGE-TAB Version 1.1 Public Release Date 2014-04-26 Investigation Title Expression analysis of an enrofloxacin and a tetracline resistant Escherichia coli strain Comment[Submitted Name] Expression analysis of an enrofloxacin and a tetracline resistant Escherichia coli strain Experiment Description Comparison of the whole genome gene expression level of an enrofloxacin and tetracycline resistant E. coli strain with the wildtype it was derived from. The process of drug adaptation of E. coli MG1655 wildtype cells is further descibed in van der Horst, M, J.M. Schuurmans, M. C. Smid, B. B. Koenders, and B. H. ter Kuile (2011) in Microb. Drug Resist. 17:141-147. Resistance to amoxicillin was induced in E. coli by growth in the presence of stepwise increasing antibiotic concentrations. To investigate consequences of the aquisition of amoxicillin resistance the transcriptomic profile of sensitive and resistant cells was compared in the absence and presence of sub-inhibitory (0.25xMIC) amoxicillin concentrations was compared. Total RNA of 3 biological replicates of E. coli MG1655 wildtype cells and drug resistant cells cultured with (0.25xMIC) or without the drug was hybridized on a 12x135k custom designed microarraychip against one common reference. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name HM-CM-$ndel HM-CM-$ndel ter Kuile Person First Name Nadine N B Person Mid Initials H Person Email geo@ncbi.nlm.nih.gov Person Affiliation University of Amsterdam Person Address Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, Amsterdam, Netherlands Person Roles submitter Protocol Name P-GSE57084-1 P-GSE57084-5 P-GSE57084-6 P-GSE57084-2 P-GSE57084-3 P-GSE57084-4 P-GSE57084-7 Protocol Description The raw data from all arrays was subjected to multiple quality control checks, i.e. visual inspection of the scans, testing against criteria for foreground and background signals, testing for consistent performance of the labeling dyes, checking for spatial effects through pseudo-color plots, and inspection of pre- and post-normalized data with box plots, ratio-intensity (RI) plots and PCA plots. Within array normalization was performed using LOESS. Between array normalization was run on the Cy3-channel by summarizing the intensity values of the probes in a probe set using the robust multi-array average (RMA) algorithm. ID_REF = VALUE = RMA values Per test sample, 5 M-5g total RNA combined with 1 M-5g random octamers (Biolegio) in 4.5 M-5l was heated to 65M-0C for 10 min to denature the RNA and was allowed to cool in an ice-waterbath for 10 min. This 4.5 M-5l was made to 10 M-5l with a first strand mastermix containing final concentrations of 50 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 75 mM KCl, 200 mM Raffinose (Sigma-Aldrich), 0.015% Triton X-100, 30 ng Actinomycin-D (Sigma-Aldrich), 0.01M DTT, 0.5 mM dGAC, 0.35 mM dUTP, 0.15 mM dUTP-Cy3 (test) or dUTP-Cy5 (common reference) (GE Healthcare) and 200U SuperScript-II (Life Technologies). This mixture was incubated for 2 min at 25M-0C, 120 min at 42M-0C and 15 min at 70M-0C. Finally, 1.5 M-5l of 2.5M NaOH was added to hydrolyze the remaining RNA by heating for 10 min at 70M-0C. 8.5 M-5l 2M MOPS was added for neutralization and the labeled cDNA was purified with the E.Z.N.A. MicroElute RNA Clean-up Kit (Omega Biotek). Dye incorporation and cDNA yield was measured on the NanoDrop ND-1000 (Thermo Scientific) yielding 2-2.5 M-5g per sample and a FOI > 10 pmol/M-5g. The common reference was made by an equimolar pool of all test samples (5 M-5g per sample) and subsequently labeled as the test samples with Cy5 incorporation. Each hybridization mixture was made up from 750 ng Test (Cy3) and 750 M-5g Reference (Cy5) sample. Samples were dried and 1.98 M-5l of water was added. The hybridization cocktail was made according to the manufacturerM-bM-^@M-^Ys instructions (Nimblegen Arrays UserM-bM-^@M-^Ys Guide M-bM-^@M-^S Gene Expression Arrays Version 5.0, Roche Nimblegen). 5.22 M-5l from this mix was added to each sample. The samples were incubated for 5 min at 65M-0C and 5 min at 42M-0C prior to loading. Hybridization samples were loaded onto 12x135k custom designed microarrays against E. coli (OID 38205, Design 120315). Microarrays were hybridized for 18 hours at 42M-0C with the NimbleGen Hybridization System 4 (Roche Nimblegen). Drug sensitive and resistant E. coli cells were grown with or without the presence of enrofloxacin or tetracycline. Treated cells were cultured with a drug concentration corresponding to 0.25xMIC (sensitive cells: 0.125 M-NM-