TTHA1939 deletion mutant strain, CS medium, 800 min after the start of inoculation
Biomaterial provider
Takushi Ooga, Osaka University
Treatment protocol
The 20 ml of culture was centrifuged by 10000 rpm, 4oC, and 5minutes. After the removal of supernatant, cell pellet was frozen by using liquid nitrogen.
Growth protocol
The T. thermophilus HB8 TTHA1939 deletion mutant strain was pre-cultured at 70oC for 6 h in 5 ml of TR medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl which was adjusted to pH 7.2 with NaOH. The cells were inoculated into 5ml of fresh TR medium while the logarithm growth phase (OD600nm = 0.5), and this inoculation was repeated again. When the OD600nm of final preculture reached 0.5, 2 ml of culture was inoculated into 200 ml fresh CS medium containing 2% Sucrose, 2% sodium glutamate, 0.055% K2HPO4, 0.018% KH2PO4, 0.2% NaCL, 0.05% (NH4)2SO4, 0.125% MgCl2 6H2O, 0.0025% CaCl2 2H2O, 0.001% FeSO4 7H2O, 0.00012% NaMoO3 2H2O, 0.00001% VOSO4 xH2O, 0.00005% MnCl2 4H2O, 0.000006% ZnSO4 7H2O, 0.0000015% 5H2O, 0.00008% CoCl2 6H2O, 0.000002% NiCl2 2H2O, 0.0001% Biotin, and 0.001% Thiamin which was adjusted to pH 7.0 with NaOH. The cells were cultured at 70oC for 800 minutes.
Extracted molecule
total RNA
Extraction protocol
Crude RNA was extracted by the addition of 1.4 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 65oC for 5 min, chilled on ice for 5 min, and then centrifuged at 4oC. 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 40 micro l of water. The RNA was treated with DNase I (Ambion, Austin, TX) at 37oC 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 70oC 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 35 units of DNase I (GE Healthcare Bio-Science Corp.) at 37oC for 10 min, and after inactivation at 98oC for 10 min, the cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturer’s instructions (ENZO Biochem. Inc., Farmingdale, NY).
Hybridization protocol
3’-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The array was incubated for 16 h at 50oC 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 GeneArray Scanner (Affymetrix).
Description
no additional information
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).
