Transcriptomic analysis of Clostridium thermocellum strain LL1210 growing in chemostat       culture at different pH values
Cells pelleted from a 45 mL sample drawn from each chemostat were         resuspended in 3 mL of TRIzol (Invitrogen, CA). A 1 ml aliquot of the TRIzol cell suspension         was used for cell lysis by bead beating with 0.8 g of 0.1mm glass beads (Biospec Products)         with 3 X 20 s bead beating treatments at 6,500?rpm in a Precellys 24 high-throughput tissue         homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). The RNA from each cell         lysate was purified and DNaseI treated using the Qiagen RNeasy kit. The RNA quantity and         quality assessed by Nanodrop and Bioanalyzer respectively. Purified RNA was depleted of rRNA         using the Ribo-Zero rRNA Removal kit for Gram Positive Bacteria (Epicentre/Illumina). The         sample was then concentrated with RNA Clean & Concentrate-5 (Zymo Research, CA)         following the manufacturer?s protocol. Depleted RNA was used as the starting material for         the Epicentre ScriptSeq? mRNA-Seq Library preparation kit (Illumina compatible) utilizing         the Epicentre Fail Safe PCR Enzyme mix (Epicentre, WI) and following the manufacturer?s         protocol. cDNA, tagged with ScriptSeq adaptors, was eluted with 20 ll of Buffer EB provided         in the Min-Elute PCR purification kit (Qiagen) according to the ScriptSeq? mRNA-Seq Library         preparation kit protocol. Twelve PCR cycles were used during library amplification and         samples were purified using the Min-Elute PCR purification kit and eluted with 20 ll of         buffer EB. The final mRNA Seq library was quantified with a Qubit Fluorometer (Invitrogen,         CA) and library quality was assessed with an Agilent Bioanalyzer High Sensitivity Chip for         DNA (Agilent, CA). Libraries were pooled and sequenced on an Illumina HiSeq 2500 platform with version 2 chemistry in a 2x151 bp configuration (JGI, Walnut Creek, CA; Project ID 503106).