alias: GSM2464879
title: GSM2464879: EFT_652_pH5.65; Fibrobacter succinogenes subsp. succinogenes S85; RNA-Seq
library_construction_protocol: Cells were pelleted from a 50 ml chemostat sample by centrifugation (7,600 × g, 4°C for 4 min), flash frozen in liquid nitrogen, and stored at -80°C. Ten milliliters of TRIzol reagent (Life Technologies) were added to the frozen cell pellet and mixed until the pellet was fully dissolved. Following the manufacturer’s protocol TRIzol was used to extract Total RNA. The aqueous phase containing RNA was further processed with a Qiagen RNeasy mini kit (Qiagen, Valencia, CA), following the manufacturer’s instructions, including the on-column DNaseI (Qiagen) treatment. Quantity of RNA was measured with NanoDrop and quality was assessed with Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) on a RNA 6000 Nano Chip.  Ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for Gram-negative Bacteria (Epicentre, Madison, WI) following the manufacturer’s protocol. RNA-seq libraries were constructed using an Epicentre Scriptseq mRNA-Seq library preparation kit (Illumina compatible).  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 RNA-seq libraries were quantified with a Qubit Fluorometer and library quality was assessed with Agilent Bioanalyzer and a DNA 7500 chip. Final libraries were normalized using the Illumina’s Library dilution calculator and pooled into a stock.  Pooled samples were prepared for sequencing based on the manufactures recomendations and sequenced on an Illumina MiSeq.
identifiers.namespace: GEO
identifiers.id: GSM2464879