Clostridium thermocellum was grown in batch fermentation to an OD600 of approximately 0.5 before treatment with either 3.95 g.L-1 ethanol, 3 g.L-1 furfural or the elevated temperature of 68°C. Total RNA was recovered at 10, 30, 60, and 120 min post-treatment for comparison with untreated controls.
Growth protocol
Batch fermentations were conducted in approximately 4 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
Briefly, samples were harvested by centrifugation and the TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total cellular RNA. Each total RNA preparation was treated with RNase-free DNase I (Ambion, Austin, TX) to digest residual chromosomal DNA and subsequently purified with the Qiagen RNeasy Mini kit in accordance with the instructions from the manufacturer. Total cellular RNA was quantified at OD260 and OD280 with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the RNA quality was checked 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
Furfural shock_Time_60
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).
