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Sample GSM2942983

Query DataSets for GSM2942983

Status

Public on Jun 19, 2020

Title

C3_5: Control Rep3_5min

Sample type

SRA

Source name

Control Rep3_5min

Organism

Caldicellulosiruptor bescii

Characteristics

tissue: Caldicellulosiruptor bescii DSM6725 cells
strain: DSM6725
setup: control
time-point: 5 min

Treatment protocol

In control fermenters, cells were grown at pH 7.2 throughout whereas, in treated fermenters, cells were grown at pH 7.2 until mid-log phase and pH was then lowered to 6.0 by acid addition.

Growth protocol

Wild-type C. bescii strain DSM6725 was grown anaerobically at 75 °C in batch fermenters with liquid LOD medium with 5 g/L maltose as the carbon source.

Extracted molecule

total RNA

Extraction protocol

Briefly, cell pellets from each sample were incubated in 250 µL of 20 mg/mL Lysozyme (Sigma Aldrich part number L-7651, St. Louis, MO) resuspended in SET buffer (50 mM Tris-HCl pH 8.0 50 mM EDTA, 20% w/v Sucrose) followed by incubation at 37oC for eight minutes, vortexing briefly every two minutes. After incubation, 900 µL of buffer RLT from a Qiagen RNeasy Kit (Qiagen, Hilden, Germany) was added to the lysozyme cell mixture and vortexed. Then 650 µL of 100% ethanol was added to each aliquot and mixed by pipetting. Each sample was then applied to a RNeasy spin column (Qiagen, Hilden, Germany) and processed following the manufacturers protocol including the optional on column Dnase I treatment. Samples were eluted in 35 µL RNAse free water (Qiagen, Hilden, Germany). The quantity and quality of RNA was assessed post-elution using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA). Ribosomal RNA was depleted from the samples using a RiboZero rRNA Removal Kit for bacteria (Illumina Inc. San Diego, CA)
cDNA libraries were prepared and barcoded using a ScriptSeq v2 RNA-Seq library preparation kit (Illumina Inc. San Diego, CA) following the manufacturers protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Control Fermenter Replicate3_5min sample (pH 7.2)

Data processing

Demultiplexing and file conversion performed by bcl2fastq version bcl2fastq-1.8.3
Sequenced reads were trimmed for adaptor sequence, masked for low-complexity or low-quality sequence and then mapped to reference genome CP001393.1 for DSM6725 strain using CLC Genomics Workbench (CLC bio, Aarhus, Denmark; version 8.0), RNA-sequencing pipeline. Default settings were used for mismatch, insertion, and deletion costs.
Differenential gene expression using the unique gene counts from CLCBio was estimated using DESeq2 .
Supplementary_files_format_and_content: Tab deliminated file includes read counts for each gene across the 30 ( 5 time-points sampled with 3 replicates each for control and treated setup, i.e. 15 per setup. Thus as two setups (control and treated ) used 15x2=30) samples used in this study

Submission date

Jan 19, 2018

Last update date

Jun 19, 2020

Contact name

Dawn Marie Klingeman

E-mail(s)

[email protected]

Phone

+18655763435

Organization name

Oak Ridge National Lab