The sample of BS_A1, BS_A2, BS_A3, BS_A4 were cultuered according to the workflow below: culturing at 37℃ for 12 hours, then taking it into 46℃ for 12 hours. It continued to culturing at 55℃ for 24 hours in the final step. The sample of BS_B1,BS_B2,BS_B3,BS_B4 were cultured at 37℃ in the whole 48 hours.
Growth protocol
A wort agar medium including a quarter of beef extract peptone was used to Bacillus subtilis fermentation for 48 hours. The seed of B.subtilis was prepared in the Luria-Bertani medium at 37℃ overnight.
Extracted molecule
total RNA
Extraction protocol
A modified CTAB method was used for total RNA extraction. The ingredient of the CTAB extraction liquid including 2% CTAB, 30mmol/L EDTA, 2mol/L NaCl, 100mmol/L Tris-HCl, pH 8.0，0.05% spermidine. adding the reagents in a clean RNase-free 1.5 ml centrifugal tube (including 600 L CTAB extraction liquid, 12 µL 2% PVP, 12 ul mercaptoethanol and final concentration of 1.5 mg/ml of proteinase K, 42 ℃ incubation for 10 minutes.Then transferred it to the sample tube and added 100 ul lysozyme (1 mg/ml), vortex oscillation, 42 ℃ incubation 90min ( vortexing several times during the time).For cell lysis nucleic acid substances released after the separation and purification of RNA as follows: in the previous step sample tube with equal volume of chloroform, isoamyl alcohol, vortex, 13000 rpm, 4 ℃ centrifuge for 15 min.Transfered the upper water phase to a new tube, repeating the above steps.Add precooling the solution (volume of 4 m LiCl and 1/2 volume of anhydrous ethanol) to the previous upper water. Precipitating the nucleic acid on ice for 3 hours. the pellets was ultraclean using 600 ul - 800 ul 2M LiCl and 75% ethanol.Obtaining total RNA.by add approprate amount DEPC water dissolved the pellets .
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
not provided
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description
BS_B3 gene experssion at a normal temperature
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
