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Sample GSM2464873

Query DataSets for GSM2464873

Status

Public on Jan 23, 2017

Title

EFT_389_pH6.7

Sample type

SRA

Source name

Bacterial fermentation

Organism

Fibrobacter succinogenes subsp. succinogenes S85

Characteristics

ph: 6.7
strain: S85

Growth protocol

Fibrobacter succinogenes S85 was grown at 37oC in a 500-ml (vessel capacity 1.3 L) culture using a water-jacketed BioFlo110 bioreactor (New Brunswick Scientific, Edison, NJ). Temperature, pH, and agitation were monitored and controlled in the bioreactor via the BioCommand 2.62 software. Cells were harvested from a 50 ml chemostat sample by centrifugation (7,600 × g, 4°C for 4 min) at each ph point.

Extracted molecule

total RNA

Extraction protocol

Cells were pelleted from a 50 ml chemostat sample by centrifugation (7,600 × g, 4°C for 4 min), flash frozen in liquid nitrogen, and stored at -80°C. Ten milliliters of TRIzol reagent (Life Technologies) were added to the frozen cell pellet and mixed until the pellet was fully dissolved. Following the manufacturer’s protocol TRIzol was used to extract Total RNA. The aqueous phase containing RNA was further processed with a Qiagen RNeasy mini kit (Qiagen, Valencia, CA), following the manufacturer’s instructions, including the on-column DNaseI (Qiagen) treatment. Quantity of RNA was measured with NanoDrop and quality was assessed with Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) on a RNA 6000 Nano Chip. Ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for Gram-negative Bacteria (Epicentre, Madison, WI) following the manufacturer’s protocol.
RNA-seq libraries were constructed using an Epicentre Scriptseq mRNA-Seq library preparation kit (Illumina compatible). Depleted RNA was used as the starting material for the Epicentre ScriptSeq™ mRNA-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™ mRNA-Seq Library preparation kit protocol. Twelve PCR cycles were used during library amplification and samples were purified using the Min-Elute PCR purification kit and eluted with 20 µl of buffer EB. The final RNA-seq libraries were quantified with a Qubit Fluorometer and library quality was assessed with Agilent Bioanalyzer and a DNA 7500 chip. Final libraries were normalized using the Illumina’s Library dilution calculator and pooled into a stock. Pooled samples were prepared for sequencing based on the manufactures recomendations and sequenced on an Illumina MiSeq.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

T389pH67_1

Data processing

Demultiplexing and file conversion performed by bcl2fastq version bcl2fastq-1.8.3
RNAseq reads were imported to CLC Genomics Workbench 6.5 (CLC bio, Boston, MA), trimmed, and then mapped to F. succinogenes S85 genome assembly (CP001792). Fastq files were imported into the CLCBio RNA-sequencing pipeline. Default settings were used for mismatch, insertion, and deletion costs.
Total gene counts from the CLCBio mapping were used for DESeq2 analysis to normalise and determine significant gene expression differences
Genome_build: ASM2466v1
Supplementary_files_format_and_content: Tab deliminated file includes read counts for each gene across the samples used in this study

Submission date

Jan 22, 2017

Last update date

May 15, 2019

Contact name

Dawn Marie Klingeman

E-mail(s)

klingemandm@ornl.gov

Phone

+18655763435

Organization name

Oak Ridge National Lab