Strains were grown in a brain-heart infusion medium (Oxoid, Hampshire, UK) at 3°C, 10°C and 37°C and sampled at the mid-exponential growth stage.
Extracted molecule
total RNA
Extraction protocol
5 ml from each sample were mixed with 1 ml of ice-cold solution containing 1 part of phenol (Sigma-Aldrich) and 9 parts of 99.5% ethanol. After 30 minutes on ice, samples were centrifuged for 5 min at 4ºC at 5000g. Bacterial pellets were immediately processed or frozen at -70ºC for later total RNA extraction. Total RNA was isolated using the Qiagen RNeasy Midi-kit (Qiagen) with slight modofications to the original kit protocol. Bacterial cells were lysed in TE buffer (Fluka Biochemica, Buchs, Switzerland) supplemented with lysozyme (25 mg/ml) and mutanolysin (250 U/ml) (Sigma-Aldrich). Two DNase treatment steps were applied to remove DNA: an initial on-column treatment with Qiagen RNase-Free DNase set (Qiagen), as well as a second DNAse treatment after isolation of total RNA using the Ambion DNA-free kit (Ambion, Austin, TX, USA). RNA yields were determined using the Nanodrop ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA), and quality was controlled using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The purified total RNA was aliquoted and frozen -70ºC for longterm storage.
Label
Cy3
Label protocol
Two micrograms of total RNA of each sample were converted to cDNA and labeled with fluorescent dyes (Cy3 or Cy5, GE Healthcare, Little Chalfont, United Kingdom).
Hybridization protocol
300 ng of labeled cDNA from each sample was hybridized onto Agilent custom 8 x 15 K DNA-microarray against equal amount of labeled control cDNA using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Three biological replicates of each strain were hybridized onto arrays, one of the replicates with a dye swap. The arrays were hybridized 16 hours at 65ºC.
Scan protocol
GenePix 4200 AL (Axon Instruments) using pixel resolution of 5 µm
Description
251575710011_5.gpr
Data processing
Image analysis with GenePix Pro 6.0, bad spots were manually flagged (flag-value -100). Data preprocessing in R with limma package: local background subtraction with normexp and offset 50. Log2-intensities normalized with quantile normalization across all samples.
