genotype: rpoN mutant (EcJR-8) growth protocol: logarithmic phase in DMEM-MOPS
Growth protocol
EcJR-8 grown to OD600 = 0.5 in DMEM-MOPS 0.4% glucose at a 10:1 flask-to-media volume in a rotary shaker (180 RPM).
Extracted molecule
total RNA
Extraction protocol
5mL of culture was mixed with 5mL of hot acid phenol:chloroform. Samples were held at 65ºC with periodic shaking for at least 10 minutes before centrifuging at 4000 rpm for 20 min. Supernatant was extracted again with acid-phenol:chloroform and then with chloroform:isoamyl alcohol (24.1). RNA was precipitated overnight at –80ºC in 2.5V 100% ethanol and 1/10V 3M sodium acetate pH 5.2. RNA samples were purified and treated with DNase using the Rneasy kit (Qiagen).
Label
Cy5
Label protocol
Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM amino-allyl dUTP. 30uL C. cDNA was purified using PCRreactions were incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
genotype: wild type growth protocol: logarithmic phase in DMEM-MOPS
Extracted molecule
total RNA
Extraction protocol
5mL of culture was mixed with 5mL of hot acid phenol:chloroform. Samples were held at 65ºC with periodic shaking for at least 10 minutes before centrifuging at 4000 rpm for 20 min. Supernatant was extracted again with acid-phenol:chloroform and then with chloroform:isoamyl alcohol (24.1). RNA was precipitated overnight at –80ºC in 2.5V 100% ethanol and 1/10V 3M sodium acetate pH 5.2. RNA samples were purified and treated with DNase using the Rneasy kit (Qiagen).
Label
Cy3
Label protocol
Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM amino-allyl dUTP. 30uL C. cDNA was purified using PCRreactions were incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
Hybridization protocol
Arrays were cross-linked by exposure to 600 mJ UV before blocking in 1% SDS, 5× SSC, and 1 mg/mL BSA at 42°C for 1 hour. After blocking, arrays were washed 2× 5 min in 0.1× SSC and 2× 30 s in H2O. Dried arrays were placed into hybridization cassettes (TeleChem International, Sunnyvale, Calif.) and the cDNA samples were suspended in 10 mM EDTA, denatured at 95°C for 5 min and then mixed with 40 uL of SlideHyb buffer 1 (Ambion) and loaded under a coverslip onto the array. Hybridizations were carried out at 47°C for 16–18 h. After hybridization, arrays were washed in 2× SSC, 0.5% SDS 37C for 5 min, followed by 2× 5 min in 0.1× SSC, 0.1% SDS 37°C, and then 2 × 2.5 min in room temperature 0.1× SSC. Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Scan protocol
Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Description
There are probes on the array that target three different E. coli genomes. Those that were specific for genomes other than Sakai were excluded from the analysis.
Data processing
The microarray data were analyzed using R (v. 2.2.1) and the MAANOVA (v. 0.98.8) package. Raw intensity values from replicate probes were averaged and log2 transformed after normalization with the pin-tip LOWESS method. The normalized intensity values were fitted to a mixed model ANOVA considering array and biological replicates as random factors and dye, strain and growth phase as fixed factors. The linear model tested was Y (intensity) = array + dye + strain (wild type or mutant) + growth phase (exponential or stationary) + strain*growth phase + sample (biological replicate) + error. Significant differences in expression due to strain, growth phase and strain*growth phase were determined using the Fs test in MAANOVA which uses a shrinkage estimator for gene-specific variance components that makes no assumption about the variances across genes with 500 random permutations to estimate the p-values. The q-value package in R was used for determining the false discovery rate (FDR).
