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Sample GSM1919124

Query DataSets for GSM1919124

Status

Public on Oct 28, 2015

Title

mRNA15, Late log phase, Xylose carbon source

Sample type

SRA

Source name

Xylose_late-log phase_whole cell

Organism

Bacteroides xylanisolvens

Characteristics

strain: XB1AT; DSM 18836T
carbon source: Xylose
growth phase: late-log phase

Growth protocol

B. xylanisolvens XB1AT (DSM 18836T) was grown anaerobically at 37°C in a complex medium containing clarified rumen fluid [33] and 5g/L of of polysaccharide (citrus peel pectin from Fluka, France; apple pectin from Sigma-Aldrich, France; oat-spelt xylan from Serva, France; washed twice in distilled water and autoclaved to remove free sugars) or sugars (glucose or xylose). The media were prepared, dispensed and inoculated by using strictly anaerobic techniques in Balch tubes. A 2.5% (v/v) inoculum of culture pre-adapted on each substrate was used for inoculation. Bacterial growth was followed by optical density of the culture at 600 nm (OD600nm) recorded directly in Balch tubes using a Jenway 6320D spectrophotometer. Three independent cultures were performed for each substrate condition for subsequent transcriptomic analyses

Extracted molecule

total RNA

Extraction 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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Illumina Casava1.8 software used for basecalling.
Quality filtering and adapter trimming were performed with Trimmomatic v0.30 using Illumina TruSeq3 adapter sequences for adapter clipping.
The B. xylanisolvens XB1A genome (GenBank accession NC_021017.1) was indexed using novoindex v1.0, and reads aligned with novoalign v. 3.00.05 (http://www.novocraft.com) against the indexed genome. For downstream gene expression analysis only the aligned R1 reads were used; these were extracted from the paired-end alignment file using samtools v0.1.19 using the bitwise flag for the first read for a read pair. Read counts were
Gene counts were determined for the aligned data using featureCounts v. 1.4.3-p1 and the NCBI GFF3 feature file (obtained from the NCBI FTP site Dec. 2013).
Differential gene expression was performed using R 3.0.0 using edgeR/limma. Samples were assessed for potential outliers based on counts using normalized counts (RPKM) that would affect downstream analyses. From this analysis we determined that samples mRNA1, 2, 3, 7 and 10 were problematic and thus removed them from the analysis. Counts per million (CPM) mapped reads were calculated per gene; genes with more 1 CPM in three or more samples were retained in the final edgeR analysis. Samples were normalized using edgeR’s TMM normalization. A simple generalized linear model was generated using the aforementioned filtered data from all remaining samples, and simple contrasts based on carbon source and growth stage were used to determine genes differentially expressed under the conditions shown.
Genome_build: B. xylanisolvens XB1A genome (GenBank accession NC_021017.1)
Supplementary_files_format_and_content: tab-delimited text, gene counts

Submission date

Oct 27, 2015

Last update date

May 15, 2019

Contact name

Christopher John Fields

E-mail(s)

cjfields@illinois.edu

Phone

217-244-1890

Organization name

University of Illinois Urbana-Champaign