TTHB211 deletion mutant strain was pre-cultured at 70oC for 16 h in 3 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 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70oC for 8 h (OD600nm = ~ 4.5).
Extracted molecule
total RNA
Extraction protocol
Cells were collected from 15 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, 5 mM EDTA, 0.25% SDS, and 50% of water-saturated phenol. 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 GeneChip Scanner 3000 (Affymetrix).
Description
TTHB211 deletion mutant strain grown on a rich medium for 480 min
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.
