Cells were collected from 10 ml of the culture with centrifugation.
Growth protocol
The T. thermophilus HB8 TTHA1559 disrupted strain was pre-cultured at 70°C until the optical density at 600 nm reached 1.0 in 10 ml of TM medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2. The cells (0.1 ml) were inoculated into 10 ml of the same medium and then cultivated at 70°C for 5 h.
Extracted molecule
total RNA
Extraction protocol
Cells were collected from 10 ml of the culture medium, and then crude RNA was extracted by the addition of 1.4 ml of a solution comprising 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.25% SDS, and 50% of TE buffer containing 10 mM Tris-HCl, pH 7.5, 1mM EDTA-saturated phenol. This mixture was incubated at 60°C for 5 min, chilled on ice for 3.5 min, and then centrifuged at 4°C. Then, 750 micro l of TRIZOL LS (Invitrogen, Carlsbad, CA) was added to 0.2 ml of the aqueous phase. After incubation for 5 min at room temperature, the RNA was extracted with 0.2 ml of chloroform. The extraction was repeated with 0.5 ml of chloroform, and the aqueous phase was precipitated with isopropanol. The pellet was dissolved in 0.2 ml of nuclease-free water, precipitated with ethanol, and then resuspended in 0.03 ml of water. The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 20 min in a 25-micro l reaction mixture. The reaction was terminated by the addition of 1 micro l of 0.5 M EDTA, followed by incubation at 70°C for 5 min. Thereafter, the RNA was precipitated with ethanol in the presence of 1% glycogen and 1 M NH4OAc.
Label
biotin
Label protocol
cDNA was synthesized with SuperScript II (Invitrogen) reverse transcriptase in the presence of RNase inhibitor SUPERase (Ambion) and 6 base random primers (Invitrogen). The cDNA was fragmented with 1.35 units of DNase I (GE Healthcare Bio-Science Corp.) at 37°C for 10 min, and after inactivation at 98°C for 10 min, the cDNA fragments were labeled with GeneChip DNA Labeling Reagent (Affymetrix, Santa Clara, CA), using terminal transferase according to the manufacturer's instructions (Affymetrix).
Hybridization protocol
3'-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401a520105F GeneChip (Affymetrix, Santa Clara, CA). The array was incubated for 16 h at 50°C in a solution comprising 100 mM MES, pH 6.6, 20 mM EDTA, 0.02% Tween 20, 7% dimethyl sulfoxide, 0.1 mg/ml of herring sperm DNA (Promega), 0.5 mg of bovine serum albumin (BSA), the recommended amount of Eukaryotic Hybridization Control (Affymetrix), and Control Oligo B2 for the alignment signal (Affymetrix), and then the array was automatically washed and stained with streptavidin-phycoerythrin (Invitrogen) by using a GeneChip Fluidics Station, 450XP (Affymetrix).
Scan protocol
The Probe Array was scanned with a GeneChip Scanner 3000 (Affymetrix).
Description
Gene expression data from TTHA1559 disrupted strain at 5h.
Data processing
The expression levels were summarized by one-step Tukey’s biweight algorithm and normalized by global scaling method using GeneChip Operating Software, version 1.4 (Affymetrix, Santa Clara, CA). The trimmed mean target intensity of each array was arbitrarily set to 500.
