title: GSM2722309: JWCB005 - mid log phase - 3; Caldicellulosiruptor bescii; RNA-Seq
library_construction_protocol: Total RNA was extracted by first incubating cell pellets in 250 µL of 20 mg/mL Lysozyme (Sigma Aldrich part number L-7651, St. Louis, MO) resuspended in SET buffer (50 mM Tris-HCl pH 8.0 50 mM EDTA, 20% w/v Sucrose) and incubated in a dry stationary bath at 37oC for eight minutes, vortexing briefly every two minutes.  900 µL of buffer RLT from a Qiagen RNeasy Kit (Qiagen, Hilden, Germany) was added and the sample was split into two equal aliquots of ~575 µL each.  325 µL of 100% ethanol was added to each aliquot and the contents were mixed by pipetting.  Contents from both tubes were spun through an RNeasy spin column (Qiagen, Hilden, Germany).  Spin columns were washed once with buffer RW1 (Qiagen, Hilden, Germany).  10 µL of DNase stock I (Qiagen, Hilden, Germany) and 70 uL of buffer RDD (Qiagen, Hilden, Germany) were combined, pipetted onto the spin column and allowed to incubate for 15 minutes at room temperature to digest residual DNA.  The columns were washed once more with buffer RW1 (Qiagen, Hilden, Germany), followed by two washes of 500 µL buffer RDE (Qiagen, Hilden, Germany) and eluted in 35 µL RNAse free H20 (Qiagen, Hilden, Germany).  RNA concentration was quantified using a Nanodrop 1000 instrument (ThermoScientific, Waltham, MA) and RNA quality was verified by obtaining RNA Integrity Numbers (RIN) using an Agilent 2100 Bioanalyzer and corresponding RNAchip (Agilent Technologies, Santa Clara, CA). 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.