GSM1671490: 0089 3-1; Hungateiclostridium thermocellum DSM 1313; RNA-Seq
Cells pelleted from a 50 mL sample drawn from each fermenter 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) utilising Epicentres Fail Safe PCR Enzyme mix (Epicentre, WI) and following the manufacturer's protocol. cDNA, tagged with ScriptSeq adaptors (1-12), was eluted with 20 µl 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 µl 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). Samples were pooled into three groups to make a stock of 20 nM in 72 ul.  A 40 ul aliquot of each of the three pooled libraries was sent to HudsonAlpha Genomic Serices Laboratory for a SR50 sequencing run on an Illumina Hiseq 2500 platform.
GEO Accession
GSM1671490