title: GSM2668674: PSR-1; Ruminiclostridium thermocellum DSM 1313; RNA-Seq
library_construction_protocol: Pelleted cells were  resuspended in TRIzol (Invitrogen, CA), throughly mixed and then refrozen.  The frozen 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 (on column) 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 Bacteria (Epicentre/Illumina). The sample was then concentrated with RNA Clean & Concentrator-5 (Zymo Research, CA) following the manufacturer’s protocol. Depleted RNA was used as the starting material for the Epicentre ScriptSeq™ v2 RNA-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 purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, IN ) and  eluted in water according to the ScriptSeq™ v2 RNA -Seq Library preparation kit protocol.  Twelve PCR cycles were used during library amplification and samples were purified with Agencourt AmPureXP magnetic beads and eluted with 20 µl of water. The final mRNA Seq library was quantified with a Qubit Fluorometer (Invitrogen, CA) and library quality was assessed with an Agilent Bioanalyzer DNA Chip (Agilent, CA). Samples were pooled and the concentration determined.  Pools of barcoded samples were sent to Hudson Alpha for SR50 sequencing run on an Illumina Hiseq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM2668674