GSM4979784: C. bescii 5′ RNA-Seq (TSS) avicel1; Caldicellulosiruptor bescii DSM 6725; RNA-Seq
Frozen cell pellets collected from 200 ml samples drawn from each fermenter were resuspended in 2 mls of TRIzol (Invitrogen, CA) and lysed by sonication at 40% power (9W) on a Misonix 3000 sonicator (Farmingdale, NY).  Samples were sonicated for a total of 15 seconds (3 x 5 second pulse followed by 1 minute off).  Chloroform was added, and the sample was mixed and centrifuged.  The aqueous layer was removed and mixed 1:1 with 80% ethanol.  Then the mixture was processed on a RNeasy column (Qiagen Hilden, Germany) following the manufactures protocol and the on-column DNase digestion.  RNA was eluted off the column in 35 µL RNAse free H20.  The RNA was quantified using a Nanodrop 1000 instrument (ThermoScientific, Waltham, MA) and RNA quality was accessed using a Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and a Nano6000 chip kit.  Purified total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal kit for Gram Positive Bacteria (Epicentre-Illumina, WI) following the manufactures protocol.  The depleted sample was concentrated on a RNA Clean & Concentrator-5 (Zymo Research, CA) following the manufacturer's protocol. 5'TSS was completed on RNASeq librareis.  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 eluted with 20 µl of Buffer EB provided in the Min-Elute PCR purification kit (Qiagen) according to the ScriptSeq™ RNA-Seq Library preparation kit protocol. Twelve PCR cycles were used during library amplification and samples were purified using Agencourt AmPureXP magnetic beads (Beckman Coulter, IN )  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 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. Libraries were prepared to investigate the 5' Transcriptional Start Site (TSS).