The 50 ml of culture was centrifuged by 9,000 x g, 4˚C, and 5 min. After the removal of supernatant, cell pellet was frozen by using liquid nitrogen.
Growth protocol
Wild-type T. thermophilus HB8 strain was pre-cultured at 70˚C for 5 h in 5 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.5 with NaOH. The cells (5 ml) were inoculated into 300 ml of the same medium and then cultivated at 70˚C. At the Abs600 =0.8 (0min), this culture was divided into two flasks. Then, equal volume of cold medium (4˚C) was added and the temperature were shifted down to 45˚C. Hot medium (70˚C) was added to the other flask. Cells were collected at 0.5 min and 10 min after temperature shift in the equal volume of 100% ice-cold ethanol, and then crude RNA was extracted.
Extracted molecule
total RNA
Extraction protocol
Crude RNA was extracted by the addition of 1.2 ml of a solution comprising 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.25% SDS, and 50% of water-saturated phenol to the cell pellet. This mixture was incubated at 65˚C for 5 min, chilled on ice for 5 min, and then centrifuged at 4˚C. Then, 0.75 ml 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 20 ml of water. The RNA was treated with DNase I (Ambion, Austin, TX) at 37˚C for 40 min in a 25-ml reaction mixture. The reaction was terminated by the addition of 1 ml 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 ammonium acetate.
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 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 biotin-dideoxy UTP, using terminal transferase according to the manufacturer’s instructions (Affymetrix).
Hybridization protocol
3’-terminal-labeled cDNA (2 mg) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The array was incubated for 16 h at 50˚C in a solution comprising 180 mM MES, pH 6.6, 40 mM EDTA, 0.02% Tween 20, 7% dimethyl sulfoxide, 1 mg 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).
Data processing
The image data was scaled to the target intensity by one-step Tukey’s biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA).
