TPGY, anaerobic, 37 °C, up to OD600=1.0 (5-7 hours), then subjected to temperature downshift to 15 °C and thereafter incubated at 15 °C.
Extracted molecule
total RNA
Extraction protocol
Five-ml samples from three replicate C. botulinum ATCC 3502 wild type cultures were collected at mid-logarithmic growth phase at 37 °C, and 1 h and 5 h after temperature downshift to 15 °C. The samples were collected into sterile plastic tubes containing ice-cold ethanol-phenol (9:1) solution (Sigma Aldrich, St. Louis, MO, USA), mixed thoroughly, and incubated on ice for 30 min. Cells were harvested by centrifugation (4 °C, 8000 x g) for 5 min. The cell pellets were immediately frozen to -70 °C until RNA extraction. The cell pellets were thawed on ice for 5 min and used for RNA extraction with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The cells were lysed with a solution containing 25 mg/ml lysozyme (Sigma Aldrich, St. Louis, MO, USA) and 250 IU/ml mutanolysin (Sigma Aldrich) in Tris-EDTA buffer (pH 8.0, Fluka BioChemica, Buchs, Switzerland) and agitated at 37 °C for 30 min. The final elution volume of RNAse free water was 300 µl. To confirm efficient removal of all genomic DNA, an additional DNase treatment was carried out using the DNA-free Kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. The RNA yield and purity (A260/A280) were checked using the NanoDrop ND-1000 device (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RNA purity ratio was >2.0 for all samples. Integrity of RNA was confirmed using a miniaturized gel electrophoresis in the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number was >9.2 for all RNA samples.
Label
Cy3
Label protocol
A total of 2 µg of each RNA sample was reverse-transcribed into cDNA and simultaneously labeled with fluorescent dyes. In brief, each 30-µl labeling reaction contained 0.2 µg/µl of random hexamers (Invitrogen), 0.01 M DTT (Invitrogen), 1.3 U/µl ribonuclease inhibitor (Invitrogen), 0.5 µM dATP, dTTP and dGTP, 0.2 µM dCTP, 1.7 nmol of Cy-3 or Cy-5 labeled dCTP (GE Healthcare, Pittsburgh, PA), 13 U/µl of SuperScript III reverse transcriptase (Invitrogen), and appropriate buffer (1 x First Strand Buffer, Invitrogen) and was incubated at 46 °C for 3 h. RNA hydrolysis and reaction inactivation were performed by addition of 10 µl of 0.1 M NaOH and 0.5 mM EDTA and incubation at 70 °C for 15 min. The reactions were subsequently neutralized by addition of 10 µl of 0.1 M HCl. The cDNA was purified with QIAquick PCR Purification Kit (Qiagen), with final elution volume of 40 µl. The cDNA concentration of each sample was measured with NanoDrop.
TPGY, anaerobic, 37 °C, up to OD600=1.0 (5-7 hours), then subjected to temperature downshift to 15 °C and thereafter incubated at 15 °C.
Extracted molecule
total RNA
Extraction protocol
Five-ml samples from three replicate C. botulinum ATCC 3502 wild type cultures were collected at mid-logarithmic growth phase at 37 °C, and 1 h and 5 h after temperature downshift to 15 °C. The samples were collected into sterile plastic tubes containing ice-cold ethanol-phenol (9:1) solution (Sigma Aldrich, St. Louis, MO, USA), mixed thoroughly, and incubated on ice for 30 min. Cells were harvested by centrifugation (4 °C, 8000 x g) for 5 min. The cell pellets were immediately frozen to -70 °C until RNA extraction. The cell pellets were thawed on ice for 5 min and used for RNA extraction with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The cells were lysed with a solution containing 25 mg/ml lysozyme (Sigma Aldrich, St. Louis, MO, USA) and 250 IU/ml mutanolysin (Sigma Aldrich) in Tris-EDTA buffer (pH 8.0, Fluka BioChemica, Buchs, Switzerland) and agitated at 37 °C for 30 min. The final elution volume of RNAse free water was 300 µl. To confirm efficient removal of all genomic DNA, an additional DNase treatment was carried out using the DNA-free Kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. The RNA yield and purity (A260/A280) were checked using the NanoDrop ND-1000 device (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RNA purity ratio was >2.0 for all samples. Integrity of RNA was confirmed using a miniaturized gel electrophoresis in the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number was >9.2 for all RNA samples.
Label
Cy5
Label protocol
A total of 2 µg of each RNA sample was reverse-transcribed into cDNA and simultaneously labeled with fluorescent dyes. In brief, each 30-µl labeling reaction contained 0.2 µg/µl of random hexamers (Invitrogen), 0.01 M DTT (Invitrogen), 1.3 U/µl ribonuclease inhibitor (Invitrogen), 0.5 µM dATP, dTTP and dGTP, 0.2 µM dCTP, 1.7 nmol of Cy-3 or Cy-5 labeled dCTP (GE Healthcare, Pittsburgh, PA), 13 U/µl of SuperScript III reverse transcriptase (Invitrogen), and appropriate buffer (1 x First Strand Buffer, Invitrogen) and was incubated at 46 °C for 3 h. RNA hydrolysis and reaction inactivation were performed by addition of 10 µl of 0.1 M NaOH and 0.5 mM EDTA and incubation at 70 °C for 15 min. The reactions were subsequently neutralized by addition of 10 µl of 0.1 M HCl. The cDNA was purified with QIAquick PCR Purification Kit (Qiagen), with final elution volume of 40 µl. The cDNA concentration of each sample was measured with NanoDrop.
Hybridization protocol
Agilent (Agilent Technologies) 8x15K CGH protocol, 65°C for 18-20 h.
Scan protocol
GenePix 4200 AL (Axon Instruments, Westburg, Leusden, The Netherlands) using pixel resolution of 5 µm, line average 2.
Description
Log ratios were not derived from a single array.
Data processing
The 37 °C and 1 h post-shock raw microarray expression data have been previously deposited in the Gene Expression Omnibus under accession number GSE26587. 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. Log2-ratio was calculated between wild type 1 h or 5 h after cold shock and wild type before cold shock and normalized with quantile normalization.
