5 mL/min in 5 mM H2SO4 using an Aminex HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA). RNA isolation and microarray analysis Fermentation samples for RNA isolation were harvested by spinning down ~30 mL culture in 50 mL Oak Ridge tubes at 8000 rpm and 4°C for 10-15 mins and the supernatant was discarded. The solid pellet fraction containing

cells and any residual Avicel® was resuspended in 1 mL of TRIzol (Invitrogen, Carlsbad, CA), flash frozen in liquid nitrogen and stored at -80°C until further use. Total RNA was extracted from the cell pellets as follows. Briefly, the frozen cell solution in TRIzol was thawed on ice and the cell solution (~1 mL) was added to a 2 mL tube containing 1 mL of 0.1 mm glass beads (BioSpec Products, Bartlesville, PLX3397 in vivo OK) ashed at 250°C overnight. Cells were lysed by rapid agitation of the tubes at 6500 rpm for 1 min in three 20s-On/20s-Off cycles using the Precellys® bead beater (Bertin Technologies, France). Subsequently, the cell lysate (~0.8 mL) in TRIzol was phase separated by addition

of 200 μL chloroform and the RNA was precipitated by addition of 500 μL 100% isopropanol. P005091 concentration The precipitated RNA pellet was washed with 1 mL of 75% ethanol and resuspended in 100 μL of RNase-free water. Any contaminating DNA was digested by in-solution DNase-I (Qiagen, Valencia, CA) treatment and the RNA sample was cleaned using the RNeasy mini kit (Qiagen, Valencia, CA) as per manufacturer’s instructions. The 6 hr time-point RNA sample was used as the reference and all other time-point samples (8, 10, 12, 14, 16 hr) were compared to the reference in cDNA/cDNA arrays. For each time-point comparison, equal amount of the extracted total RNA samples was labeled with Cy3-dUTP/Cy5-dUTP fluorescent dyes (GE Healthcare, Piscataway, NJ), mixed and hybridized

onto custom oligo-arrays in dye swap experiments as described earlier [17] and microarray slides were scanned in ScanArray Express scanner (Perkin Elmer, Waltham, MA). Microarray construction and statistical data analysis Microarrays containing 2980 unique and 10 group 70-mer oligonucleotide probes representing ~97% of the 3163 Open Reading Frames (ORFs) this website in the draft assembly of C. thermocellum ATCC 27405 were constructed as described earlier [15]. The probe sequences were later compared to the completed genome sequence using reciprocal BLAST analysis and assigned new ORF numbers. Based on the comparison, 79 probes which did not have any BLAST hits and 108 probes that only had partial hits to annotated ORFs in the closed genome were either excluded or marked-up during downstream data analysis. Signals were quantified in ImaGene version 6.0 (BioDiscovery Inc., El Segundo, CA) and statistical data analysis was conducted using JMP Genomics software (SAS Institute Inc., Cary, NC). The array signal intensities were background-corrected, log2-transformed and data for duplicated probes on the arrays were averaged and normalized using the Data-Standardize method.