Continuously grown C. botulinum was subjected to heat shock at 45°C and the growth was continued at this temperature.
Growth protocol
C. botulinum was continuously grown in buffered tryptone-peptone-glucose-yeast-extract broth in a fermenter at 39°C under anaerobic conditions at a controlled pH of 6.8.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Qiagen's RNeasy Midi Kit (Qiagen, Hilden, Germany), with on-column DNase treatment, following manufacturer's instructions. Additional DNase treatment using the Ambion DNA-free kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
2 μg total RNA was reverse transcribed and labeled directly with fluorescent dye for 3 h at 46˚C. The mixture contained 5 μg of random primers, 40 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, 6 μl 5x first-strand buffer, 3 μl of 100 mM DTT, 400 U SuperScript™ III Reverse Transcriptase (all Invitrogen, Life Technologies Ltd, Paisley, UK), 0.6 μl dNTP mix (25 mM dATP, 25 mM dGTP, 25 mM dTTP, 10 mM dCTP [Promega Corporation, Madison, WI, USA]), and 2 nmol Cy3-dCTP or Cy5-dCTP (GE Healthcare, Buckinghamshire, UK). The labeled cDNA was purified using a DNA purification column (QIAquick PCR purification kit; Qiagen).
Continuously grown C. botulinum was subjected to heat shock at 45°C and the growth was continued at this temperature.
Growth protocol
C. botulinum was continuously grown in buffered tryptone-peptone-glucose-yeast-extract broth in a fermenter at 39°C under anaerobic conditions at a controlled pH of 6.8.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Qiagen's RNeasy Midi Kit (Qiagen, Hilden, Germany), with on-column DNase treatment, following manufacturer's instructions. Additional DNase treatment using the Ambion DNA-free kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
2 μg total RNA was reverse transcribed and labeled directly with fluorescent dye for 3 h at 46˚C. The mixture contained 5 μg of random primers, 40 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, 6 μl 5x first-strand buffer, 3 μl of 100 mM DTT, 400 U SuperScript™ III Reverse Transcriptase (all Invitrogen, Life Technologies Ltd, Paisley, UK), 0.6 μl dNTP mix (25 mM dATP, 25 mM dGTP, 25 mM dTTP, 10 mM dCTP [Promega Corporation, Madison, WI, USA]), and 2 nmol Cy3-dCTP or Cy5-dCTP (GE Healthcare, Buckinghamshire, UK). The labeled cDNA was purified using a DNA purification column (QIAquick PCR purification kit; Qiagen).
Hybridization protocol
300 ng of Cy3-labeled and of 300 ng of Cy5-labeld cDNA samples were mixed with 2.3 μg of salmon sperm DNA (Invitrogen), denaturized for 2 min at 95°C, then mixed with 5 μl 10x blocking agent and 26 μl 2x RPMI hybridization buffer (Agilent) and hybridized for 16 h at 65°C. After hybridization the slides were washed according to the manufacturer’s instructions.
Scan protocol
GenePix 4200 AL (Axon Instruments) using pixel resolution of 5 µm, line average 2
Description
technical replicate (dye-swap) of 2. 18 h
Data processing
Image analysis with GenePix Pro 6.0, bad spots were manually flagged (flag-value -100). Data preprocessing in R with limma package: background subtraction with normexp and offset 50. Loess-normalization of log2(Cy5/Cy3).
