Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
normalization data transformation protocol
GeneSpring software (Agilent) was used to compare groups of triplicate datasets for (i) wild-type cells grown with vs without arabinose, (ii) wild-type vs delta araC cells grown in the presence arabinose, and (iii) wild-type vs delta araC cells grown in the absence of arabinose. Analysis incorporates replicates. Each value is a comparison of 3 replicates for one strain/condition to 3 replicates for another strain/condition.
nucleic acid labeling protocol
cDNA synthesis, labeling, hybridization to Affymetrix GeneChip E. coli Genome 2.0 microarrays, washing and scanning were performed according to the manufacturer’s (Affymetrix) instructions
In some cases cells were grown with 0.2% arabinose throughout the culture growth
nucleic acid extraction protocol
1 ml cells were mixed with 400 ul ice-cold 95% ethanol and 5% phenol/chloroform/isoamyl alcohol (25:24:21 mix). Cells were pelleted in a microcentrifuge for 1 minute at full speed and washed once with TBS. Cell pellets were resuspended in 400 ul RNA lysis buffer (2% SDS, 4 mM EDTA) and boiled for 3 minutes. 400 ul acid phenol/choroform/isoamyl alcohol mix (pH 4.3) was added and incubated at 65 degrees C for 6 minutes and on ice for 5 minutes. Samples were centrifuged and the aqueous layer was extracted once more with phenol/choroform/isoamyl alcohol mix (pH 4.3). RNA was precipitated with 1 ml 100% ethanol and 40 ul 3 M sodium acetate. RNA was pelleted in a microcentrifuge for 10 minutes at full speed and washed once with room-temperature 75% ethanol. RNA pellets were air-dried and resuspended in water and treated with 10 units of DNase I (NEB) in 500 ul for 1 hour at 37 degrees C. RNA was then phenol extracted and ethanol precipitated. For each sample we generated three independent biological replicates labeled B, C or D.
RNA was purified from wild-type MG1655 or MG1655 delta araC cells grown in LB at 37 degrees C to an OD600 of 0.6-0.8.