GSM1251936: DSM 1237 exponential replicate 2; Hungateiclostridium thermocellum; RNA-Seq
Cells for RNAseq sampling were grown in anaerobic Balch tubes (26 mL; Bellco Glass Inc., Vineland, NJ) in 10 mL of 1191 medium (pH 7.2) on 2.2 g L-1 cellobiose.  1191 media preparation and inoculation protocols were followed as described by Islam et al.  Samples were taken in exponential and stationary phase (OD600 ~ 0.37 and ~0.80, respectively).  Cell pellets were stored at -80 °C in 1 ml RNAlater until ready for processing. Total RNA was extracted from 10 ml mid-exponential and late exponential  (OD600 ~ 0.37 and ~0.80, respectively) Clostridium thermocellum cultures using the RNeasy Mini kit (QIAGEN, Valencia, CA, USA) protocol as described for gram-positive bacteria, but with the following modifications.  Cell pellets were collected by centrifugation and re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 20 mg/ml lysozyme and incubated at 37 °C for 30 minutes.  To ensure efficient lysis, a mechanical shearing step was included that involved passing the cell pellets 10 times through a 22G syringe prior to the addition of ethanol.  Total RNA was eluted in 30 μl RNase-free water.  Following the first elution, the entire volume was re-applied to the mini column, incubated for 3 minutes and finally centrifuged at 8000 x g for 1 minute. Illumina Truseq mRNA
GEO Accession
GSM1251936