In the case of TTHA0137, TTHA0540 and TTHA1817, an equal volume of hot medium was added to the culture. In the other disruptants, cultures were added to an equal volume of 100% ethanol and stored at -80°C.
Growth protocol
The T. thermophilus HB8 wild-type strain and disruptants were cultured in TT medium at 70°C until the cells reached to OD600 of 0.8 or to stationary phase.
Extracted molecule
total RNA
Extraction protocol
Collected cells were mixed with 1.4 ml of 5 mM Tris-HCl (pH 7.5) containing 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. Aqueous phase (0.2 ml) was collected and mixed with 0.75 ml of TRIZOL LS (Invitrogen, Carlsbad, CA), and incubated at room temperature for 5 min. Then, 0.2 ml of chloroform was added, and the mixture was vortexed for 10 sec. After centrifugation, aqueous phase was collected, mixed with 0.5 ml of chloroform, and vortexed for 10 min. The aqueous phase was collected and mixed with 0.5 ml of isopropanol to precipitate RNA. After incubation at -20°C for 60 min and centrifugation, aqueous phase was removed. The pellet was dissolved in 0.2 ml of nuclease-free water, and further purified through the ethanol precipitation, and then resuspended in 0.2 ml of water. The obtained RNA solution was treated with DNase I (Ambion, Austin, TX) at 37°C for 20 min in a 25 μl reaction mixture. The reaction was terminated by the addition of 1 μl 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
10 μg of total RNA was mixed with 750 ng of random primers (Invitrogen), incubated at 70°C for 10 min, and 25°C for 10 min. The RNA/primer solution was reacted with SuperScript II reverse transcriptase (Invitrogen) to synthesize cDNA. The reaction was performed in the presence of RNase inhibitor SUPERase (Ambion) and 6 base random primers (Invitrogen). The synthesized DNA was purified by ethanol precipitation, and 3 μg of cDNA was fragmented with 35 units of DNase I (GE Healthcare) at 37°C for 10 min. The reaction was stopped by incubation of the reaction solution at 98°C for 10 min. 3 μg of fragmented cDNA was labeled with GeneChip DNA Labeling Reagent (Affymetrix), using terminal transferase according to the manufacturer’s instructions (Affymetrix). Gel-shift assay using NeutrAvidin (Invitrogen) as a probe confirmed that the majority of cDNA was successfully labeled with biotin.
Hybridization protocol
2 μg of 3’-terminal-labeled cDNA was hybridized to a TTHB8401a520105F GeneChip (Affymetrix). 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, the recommended amount of Eukaryotic Hybridization Control (Affymetrix), and Control Oligo B2 for the alignment signal (Affymetrix). Then the array was automatically washed and stained with streptavidin, biotin anti-streptavidin, and 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
cultures were added to an equal volume of 100% ethanol and stored at -80°C.
Data processing
The image data was scaled to the target intensity by one-step Tukey’s biweight algorithm using GeneChip Operating software, version 1.4 (Affymetrix). Scaled probe value was calculated as Sc = 500 according to Statistical Algorithms Description Document (Affymetrix).
