batch fermentations with: Populus time (hours): 12
Treatment protocol
Clostridium thermocellum was grown in batch fermentations with populus or switchgrass. Total RNA was recovered at 12 and 37 hours for comparison.
Growth protocol
Batch fermentations were conducted in approximately 5 L of MTC medium in 7.5 L BioFlo110 biopreactors (New Brunswick Scientific, Edison, NJ) fitted with agitation, pH and temperature probes and controls as described previously (Yang et al., 2008).
Extracted molecule
total RNA
Extraction protocol
Cells were harvested by centrifugation and resuspended in Trizol reagent (Life Technologies, CA). The suspension was transferred to a 2.0 mL screw top tube containing 800 mg of ashed glass beads. Trizol was added to make up any remaining volume in the tube and then each tube was disrupted for three 20 second bead beating treatments at 6,500 rpm in a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Chloroform was added and mixed with the sample, and then separated by centrifugation. The aqueous layer was collected in fresh tubes and a 1:1 volume of 75% ethanol was added. After mixing, the sample was applied to an RNeasy column (Qiagen, CA) and processed following the manufacturer’s instructions including an on column DNA digestion. Total cellular RNA was quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE) and RNA quality was assessed with Agilent Bioanalyzer (Agilent, CA). The purified RNA with good quality from each sample was used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit (Invitrogen, CA).
Label
Cy3
Label protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Hybridization protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Scan protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Description
F185_Pop_12 hr rep1
Data processing
Statistical analysis was done with JMP Genomics 4.0 software (SAS Institute, Cary, NC). The data were subsequently normalized using the Loess normalization algorithm within JMP Genomics. An analysis of variance (ANOVA) was performed to determine differential expression levels between conditions and time points using the FDR testing method (p < 0.05).
