Title: Affymetrix CEL analysis. Description:
The signal intensities of all probes for genes in E. coli K-12 that are annotated as genes in MG1655 (the strain of E. coli K-12) by Affymetrix (4070 genes described as MG1655_ in the description for the Affymetrix E. coli genome 2.0 array) were adjusted to confer the signal average (the trimmed mean signal) to 500 (the target signal) for each array, using the scaling function of GCOS software supplied by Affymetrix (USA). The trimmed mean signal, that is the average value of all observations in each array (in this analysis, all observations mean all signal intensities of probes corresponding to the genes in K-12) after removing the values in the lowest 2% and upper 2% of all observations, was automatically computed by GCOS. The 500 is the default value as the target signal for the scaling function of GCOS.
Synthesis of cDNA, terminal labeling, and hybridization with the oligonucleotide chip were all performed following the Affymetrix instruction manual (Affymetrix, USA). The utilized cDNA was synthesized from 10 ?g of total RNA using random primers and Superscript III reverse transcriptase (Invitrogen, Norway), followed by purification using QIAquick purification columns (Qiagen, Germany) and digestion with DNaseI (GE Healthcare, UK). The cDNA fragments were labeled with biotin-ddUTP using the GeneChip DNA Labeling Reagent (Affymetrix, USA).
(Parameters: Amount of nucleic acid labeled = 10, Amplification = none, Mass unit = Micro gram)
This protocol is the standard protocol supplied by Affymetrix.
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 3, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 45)
E. coli strains were cultivated in LB medium (10 g of Bacto Trypton and 5 g of Yeast Extract [DIFCO, USA] per liter) supplemented with 0.3 M NaCl (final concentration). W3110, W3110 hha::Km (TU04), W3110 ydgT::Cm (TU11), W3110 hha::Km ydgT::Cm (TU07), W3110 hns::Km (TU03), W3110 stpA::Cm (ZEU02) and W3110 hns::Km stpA::Cm (TU05) cells were grown in 10 ml of LB (+0.3 M NaCl) medium under aerobic conditions at 37°C until the culture reached an OD600 of 0.4.
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = LB medium (10 g of Bacto Trypton and 5 g of Yeast Extract [DIFCO, USA] per liter) supplemented with 0.3 M NaCl (final concentration))
The 5 ml of culture was mixed with 10 ml of RNA Protect (Qiagen, Germany) and the cells were collected by centrifugation and stored at ?80°C. Total RNA was purified from collected cells using the RNeasy mini kit according to the manufacturer's instructions (Qiagen, Germany).
(Parameters: Extracted product = total_RNA, Amplification = none)