Portions of 40-ml of the pre-culture of B. subtilis were exposed to seven different temperature regimes in 250-ml bottles in a reciprocal-shaking water bath at 65ºC. Duplicate samples were taken when the temperature in the bottles reached 40, 50, 57, 58, 59, 60, and 62ºC (at incubation times of approximately 100, 200, 300, 320, 380, 500 and 720 s, respectively).
Growth protocol
An overnight culture of B. subtilis was diluted 20x in TSB to a volume of 600 ml and incubated at 30°C at 100 rpm till OD600 reached 0.5.
Extracted molecule
total RNA
Extraction protocol
Immediate quenching of metabolic activity in the samples was performed with cold methanol, according to described methods. Briefly, 40-ml culture samples were sprayed in 160-ml of a stirred solution of 60% methanol, 66.7 mM HEPES, pH 6.5 that was kept at -45ºC in an ethanol bath. Subsequently, cells were disrupted in a 2-ml screw-cap tube in the presence of 100-um zirconium beads and SDS/phenol for 60 seconds in the Minibeadbeater 8TM (Biospec Products). After chloroform extraction, nucleic acids were precipitated, vacuum-dried, dissolved in distilled water and incubated for 15 min in the presence of DNase I (Qiagen). Finally, RNA was purified by phenol/chloroform extraction and ethanol precipitation as verified by absorption properties in the 200-300 nm region by the use of the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA).
Label
Cy5
Label protocol
Fluorescently labeled cDNA was prepared from 12.5 μg of total RNA by random hexamer pd(N)6-primer (Roche, Mannheim, Germany) polymerization using Superscript II reverse transcriptase (Life Technologies). Concentrations of nucleotides in labeling reaction mixture were 0.4 mM dATP, dGTP, dCTP and 0.2 mM dTTP. The final concentration of Cy3-dUTP or Cy5-dUTP (GE Healthcare) was 0.1 mM. Unincorporated Cy-dye-labeled dUTP, dNTP’s, primers and salts were removed by purification with AutoSeq G50 columns (GE Healthcare).
Portions of 40-ml of the pre-culture of B. subtilis were exposed to seven different temperature regimes in 250-ml bottles in a reciprocal-shaking water bath at 65ºC. Duplicate samples were taken when the temperature in the bottles reached 40, 50, 57, 58, 59, 60, and 62ºC (at incubation times of approximately 100, 200, 300, 320, 380, 500 and 720 s, respectively).
Growth protocol
An overnight culture of B. subtilis was diluted 20x in TSB to a volume of 600 ml and incubated at 30°C at 100 rpm till OD600 reached 0.5.
Extracted molecule
total RNA
Extraction protocol
Immediate quenching of metabolic activity in the samples was performed with cold methanol, according to described methods. Briefly, 40-ml culture samples were sprayed in 160-ml of a stirred solution of 60% methanol, 66.7 mM HEPES, pH 6.5 that was kept at -45ºC in an ethanol bath. Subsequently, cells were disrupted in a 2-ml screw-cap tube in the presence of 100-um zirconium beads and SDS/phenol for 60 seconds in the Minibeadbeater 8TM (Biospec Products). After chloroform extraction, nucleic acids were precipitated, vacuum-dried, dissolved in distilled water and incubated for 15 min in the presence of DNase I (Qiagen). Finally, RNA was purified by phenol/chloroform extraction and ethanol precipitation as verified by absorption properties in the 200-300 nm region by the use of the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA).
Label
Cy3
Label protocol
Fluorescently labeled cDNA was prepared from 12.5 μg of total RNA by random hexamer pd(N)6-primer (Roche, Mannheim, Germany) polymerization using Superscript II reverse transcriptase (Life Technologies). Concentrations of nucleotides in labeling reaction mixture were 0.4 mM dATP, dGTP, dCTP and 0.2 mM dTTP. The final concentration of Cy3-dUTP or Cy5-dUTP (GE Healthcare) was 0.1 mM. Unincorporated Cy-dye-labeled dUTP, dNTP’s, primers and salts were removed by purification with AutoSeq G50 columns (GE Healthcare).
Hybridization protocol
Microarray slides were incubated for 45 min at 42ºC with prehybridization solution (1% bovine serum albumin, 5x SCC and 0.1% SDS, filtered), washed three times with milliQ water, and dried by the use of a nitrogen flow. The Cy3-labeled cDNA from untreated cells sampled at 30ºC, was mixed with Cy5-labeled cDNA from heat-treated cells for each hybridization. After 2 min denaturation at 95 ºC, hybridizations of microarray slides were performed overnight at 42ºC in 40 μl of EasyHyb buffer (BioCat, Heidelberg, Germany) with both labeled cDNAs and 2.5 mg ml-1 yeast tRNA (final concentration). Microarray slides were washed at room temperature for 10 sec in 1x SSC/0.2% SDS at 37 ºC, 10 sec in 0.5x SSC at 37 ºC, and twice for 10 min in 0.2x SSC at room temperature. Slides were dried by the use of a nitrogen flow.
Scan protocol
ch1: laser power(%)/sensitivity(%): 90 / 75 ch2: laser power(%)/sensitivity(%): 100 / 70 Slides were scanned with a ScanArray 5000 laser scanner (Perkin Elmer Life Sciences). The TIFF-images were quantified with the software package ImaGene version 5.6.1 (BioDiscovery, El Segundo, CA, USA).
Description
Bsu1A1_59deg_rep2
Data processing
Data were processed by the use of Microsoft Office Excel 2003 SP2. The intensities of 8200 spots were calculated by extraction of the median background (B) from the median signal (S) and values for S-B < 1 were set to 1. The raw Cy5/Cy3 ratios (r) were calculated from the remaining background-corrected median signals. The total intensity normalization was carried out by the normalization factor N, defined as the average Cy5 (S-B) divided by the average Cy3 (S-B)). The normalization factor was determined from spot intensities with S/B > 2 for both Cy5 and Cy3 channels in order to avoid bias resulting from the degradation of the major of the transcripts at temperatures from 57-62°C. The normalised Cy5/Cy3 ratios (R=r/N) with S/B > 2 for either the Cy5 or Cy3 channel were calculated. Ratios from duplicate spots on the array and 2 independent experiments were averaged and 2log transformed.
