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Sample GSM1671489

Query DataSets for GSM1671489

Status

Public on Oct 31, 2015

Title

0089 2-1

Sample type

SRA

Source name

Bacterial fermentation

Organism

Acetivibrio thermocellus DSM 1313

Characteristics

strain: delta-hpt;delta-Clo1313_0089
time point sampled: Mid-exponential growth phase

Growth protocol

Strains of Clostridium thermocellum were grown in pH controlled 1L Q-Plus bioreactors (Sartorius) using MTC growth medium and cellobiose (5g/L) as the substrate (58°C, 100 rpm). Cells were harvested from 50 ml samples taken from the fermenter at mid-exponential, late exponential and stationary growth phases. Each fermentation was performed in triplicate.

Extracted molecule

total RNA

Extraction protocol

Cells pelleted from a 50 mL sample drawn from each fermenter were resuspended in 3 mL of TRIzol (Invitrogen, CA). A 1 ml aliquot of the TRIzol cell suspension was used for cell lysis by bead beating with 0.8 g of 0.1mm glass beads (Biospec Products) with 3 X 20 s bead beating treatments at 6,500 rpm in a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). The RNA from each cell lysate was purified and DNaseI treated using the Qiagen RNeasy kit. The RNA quantity and quality assessed by Nanodrop and Bioanalyzer respectively. Purified RNA was depleted of rRNA using the Ribo-Zero rRNA Removal kit for Gram Positive Bacteria (Epicentre/Illumina). The sample was then concentrated with RNA Clean & Concentrate-5 (Zymo Research, CA) following the manufacturer’s protocol.
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 mRNA Seq library was quantified with a Qubit Fluorometer (Invitrogen, CA) and library quality was assessed with an Agilent Bioanalyzer High Sensitivity Chip for DNA (Agilent, CA). Samples were pooled into three groups to make a stock of 20 nM in 72 ul. A 40 ul aliquot of each of the three pooled libraries was sent to HudsonAlpha Genomic Serices Laboratory for a SR50 sequencing run on an Illumina Hiseq 2500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

SL92474

Data processing

Demultiplexing and file conversion performed by bcl2fastq version bcl2fastq-1.8.3
RNAseq reads were aligned to the C. thermocellum DSM1313 (NC_017304.1) genome assembly using Fastq files 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
Supplementary_files_format_and_content: Tab deliminated file includes read counts for each gene across the 36 samples used in this study

Submission date

Apr 29, 2015

Last update date

May 15, 2019

Contact name

Charlotte Wilson

Organization name

ORNL

Department

Biosciences Bldg 1520

Lab

329