alias: GSM1919114
title: GSM1919114: mRNA5, Late log phase, Citrus Pectin carbon source; Bacteroides xylanisolvens; RNA-Seq
library_construction_protocol: Total RNAs were isolated from cultures harvested at mid- and late-log phase using a modified guanidinium–phenol–chloroform procedure previously described for rumen fluid. Briefly, bacterial cultures (4 tubes x 8 ml) were centrifuged for 15 min at 3,000 g at 4 °C. The pellets were resuspended in 9 ml of a RNA-E solution containing solution D [39], water saturated phenol, sodium acetate 0.2 M pH 4.0 and 2-mercaptoethanol (1:1:0.1:0.007). Cells were then disrupted by bead beating for 1 min with 0.1 g zirconia beads (0.1 mm) followed by a 2-min incubation at 60 °C. These two steps were then repeated once. After addition of 3.75 ml of chloroform, the samples were briefly mixed, incubated for 15 min on ice and centrifuged (12,000 g, 20 min, 10 °C). The RNAs contained in the aqueous supernatants (approximately 6 ml) were precipitated with 0.25 volume isopropanol and washed with 1 volume 75% cold ethanol in DEPC-treated water. Total RNAs were solubilized in 100 µl of DEPC-treated water. Genomic DNA was removed using the Turbo DNA-Free DNAse (Ambion, France) for 30 min at 37°C. RNAs were quantified using a ND-2000 NanoDrop spectrophotometer (Nanodrop Technologies, France). Enriched fractions of mRNAs were prepared using the MicrobExpress™ Bacterial mRNA Purification kit (Ambion, France). The high RNA quality and the reduction in 16S and 23S rRNA in enriched fractions of mRNAs were confirmed using an Agilent 2100 Bioanalyser (Agilent technologies, France).  cDNA libraries were prepared with 100ng of mRNA-enriched fractions following the protocols of the Illumina TruSeq Stranded RNA-seq Sample Preparation Kit. The final libraries had an average fragment size of ∼250 bp and were quantified by qPCR before being sequenced with an Illumina HiSeq 2000 instrument on a single lane in paired end reads
identifiers.namespace: GEO
identifiers.id: GSM1919114