Originally purchased from American Type Culture Collection (Manassas, VA, USA).
Treatment protocol
A RNA pool was generated by combining 25 microg of RNA from isolations from samples taken from the Bioflow II reactor at 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66 hrs after bioreactor inoculation.
Growth protocol
Colonies of C. acetobutylicum ATCC 824 (American Type Culture Collection, Manassas, VA, USA) were grown on agar-solidifed 2xYTG plates and used to start preinoculum cultures as previously described (Alsaker et al. 2005. Biotechnology and Bioprocess Engineering 10: 432-443). Preinoculum cultures were grown to OD(600 nm) of 0.2 and transferred to a 3.6 liter working volume reactors (Bioflow II and Bioflow 110, New Brunswick Scientific, Edison, NJ) in a 10-fold dilution of CGM. The fermentation was pH controlled at a low end of 5.0 with 6N NH4OH. Anaerobic conditions were maintained with nitrogen at flow rates of 125 ml/min (for an OD(600 nm) below 1.0) and 25 ml/min (for an OD(600 nm) above 1.0).
Extracted molecule
total RNA
Extraction protocol
Cell pellets were collected by centrifuging 3 to 10 mL of cultures at 5,000xg for 10 min at 4 degrees C, and stored at -80 degrees C until the RNA was isolated. Immediately prior to RNA isolation, cells were washed in 1 mL of SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0]) and collected by centrifuging at 5,000xg for 10 min at 4 degrees C. Cell pellets were then suspended in 220 microL SET buffer containing 20 mg/mL lysozyme (Sigma, St. Louis, MO, USA) and 4.55 U/ml proteinase K (Roche Applied Science, Indianapolis, IN, USA). The sample was incubated at room temperature for 6 min followed by 4 min continuous vortexing at room temperature. 1 mL of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added and the sample was vortexed. 500 microL of sample was diluted with an equal amount of ice cold TRIzol and then purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions (RNeasy Lipid Tissue Handbook, Qiagen); buffer RW1 was incubated for 4 minutes at room temperature to minimize genomic DNA contamination. Isolated RNA was eluted in 30-40 microL RNase-free water and quantified using a spectrophotometer by measuring absorbance at 260 and 280 nm. Samples were only used if the A260/A280 ratio was greater than 1.8 and RNA integrity was verified by running 0.5 microL of sample on a 1.0% agarose gel. Purified RNA was stored at -85 degrees C and used within one week of purification.
Label
Cy3
Label protocol
An indirect labeling approach was followed where cDNA was first made in the presence of nucleotides and aminoallyl dUTP, purified, and then the aminoallyl dUTP groups were coupled to a monoreactive dye (Cy3 or Cy5) as described previously (Alsaker et al. 2005. Biotechnology and Bioprocess Engineering 10: 432-443).
Channel 2
Source name
Clostridium acetobutylicum ATCC 824 time course sample taken 44 hours after bioreactor inoculation.
Originally purchased from American Type Culture Collection (Manassas, VA, USA).
Treatment protocol
Sample taken 44 hours after bioreactor inoculation.
Growth protocol
Same as channel 1
Extracted molecule
total RNA
Extraction protocol
Same as channel 1
Label
Cy5
Label protocol
Same as channel 1
Hybridization protocol
Arrays were hybridized according to Agilent’s Hybridization Procedure for 22k microarrays (cDNA labeled Targets) (Agilent 60-mer Oligo Microarray Processing Protocol v. 4.1, Agilent); however, the 17 hr incubation was performed at 65 degrees C. Washing of the arrays was performed according to Agilent’s Wash Procedure with Stabilization and Drying Solution (Two-Color Microarray-Based Gene Expression Analysis v. 4.0.1, Agilent). Removal of microarrays from the final Stabilization and Drying Solution wash was immediately repeated for slides that were not completely dry.
Scan protocol
Scanned using a G2565BA Agilent Microarray Scanner (Agilent, Wilmington, DE, USA) at 10 micrometers resolution and PMTs were set to 60 and 10.
Description
No additional information.
Data processing
Microarrays were scanned using a G2565BA Agilent Microarray Scanner (Agilent) at 10 microm resolution. Scans were performed using Agilent’s eXtended Dynamic Range technique. Each array was scanned twice: once at photomultiplier tube gain of 100%, and again at a 10% gain. Information from both scans was processed by Agilent Feature Extraction software (v. 9.1), used to assess spot quality and obtain feature intensity statistics; duplicate spots were averaged. Background-subtracted data were normalized using a segmental nearest neighbor logarithmic expression ratio-of-the mean (SNN-LERM) approach (Yang et al., PNAS 2003; 100:1122-1127).
