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Sample GSM2722312

Query DataSets for GSM2722312

Status

Public on Jul 30, 2018

Title

JWCB005 - late log phase - 3

Sample type

SRA

Source name

Caldicellulosiruptor bescii strain JWCB005

Organism

Caldicellulosiruptor bescii

Characteristics

stage of log phase: late
genotype: delta_pyrF
background strain: JWCB005

Treatment protocol

Strain JWCB005 was the genetic parent strain to strain JWCB005delta_rex. JWCB005delta_rex was the treatment while JWCB005 was the control. JWCB005delta_rex contains a markerless deletion of the rex gene (ATHE_RS03255).

Growth protocol

Cells were cultured in 3L fermenters in a working/culture volume of 1.5 L at a growth temperature of 75oC. Maltose, resazurin and an appropriate volume of water were added to assembled fermenters and autoclaved. Fermenters were allowed to cool while sparging with N2 gas. Upon cooling, the other media components were added as pre-sterilized stock solutions. The media was again heated and sparged with N2 gas. Upon reaching temperature, the pH was adjusted to 7.1 by sparging 80%/20% N2/CO2. The pH was then aseptically checked using a second probe which had been calibrated with fresh pH buffers maintained at 75oC. Any adjustments differences were inputted as the pH probe offset. Fermenters were inoculated to equivalent OD680’s of 0.01-0.05 using batch grown cultures grown to mid-log phase. Stirring was maintained at 200 rpm, and no gas was sparged during growth, though the outlet line was kept open to allow fermentation off-gasses to vent through a sterilized water trap.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted by first incubating cell pellets 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) and incubated in a dry stationary bath at 37oC for eight minutes, vortexing briefly every two minutes. 900 µL of buffer RLT from a Qiagen RNeasy Kit (Qiagen, Hilden, Germany) was added and the sample was split into two equal aliquots of ~575 µL each. 325 µL of 100% ethanol was added to each aliquot and the contents were mixed by pipetting. Contents from both tubes were spun through an RNeasy spin column (Qiagen, Hilden, Germany). Spin columns were washed once with buffer RW1 (Qiagen, Hilden, Germany). 10 µL of DNAse stock I (Qiagen, Hilden, Germany) and 70 uL of buffer RDD (Qiagen, Hilden, Germany) were combined, pipetted onto the spin column and allowed to incubate for 15 minutes at room temperature to digest residual DNA. The columns were washed once more with buffer RW1 (Qiagen, Hilden, Germany), followed by two washes of 500 µL buffer RDE (Qiagen, Hilden, Germany) and eluted in 35 µL RNAse free H20 (Qiagen, Hilden, Germany). RNA concentration was quantified using a Nanodrop 1000 instrument (ThermoScientific, Waltham, MA) and RNA quality was verified by obtaining RNA Integrity Numbers (RIN) using an Agilent 2100 Bioanalyzer and corresponding RNAchip (Agilent Technologies, Santa Clara, CA).
Depleted RNA was used as the starting material for the Epicentre ScriptSeq™ v2 RNA-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 purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, IN ) and eluted in water according to the ScriptSeq™ v2 RNA -Seq Library preparation kit protocol. Twelve PCR cycles were used during library amplification and samples were purified with Agencourt AmPureXP magnetic beads and eluted with 20 µl of water. The final mRNA Seq library was quantified with a Qubit Fluorometer (Invitrogen, CA) and library quality was assessed with an Agilent Bioanalyzer DNA Chip (Agilent, CA). Samples were pooled and the concentration determined. Pools of barcoded samples were sent to Hudson Alpha for SR50 sequencing run on an Illumina Hiseq 2500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Reads obtained were scored for quality, trimmed, mapped, and the mapped reads counted using the corresponding functions in the CLC Genomics Workbench version 8 using default settings for genome analysis of prokaryotes.
Raw read counts for each coding sequence were used as input for differential expression analysis using the DEseq2 package as part of the Bioconductor Suite in R
Genome_build: NC_012034
Supplementary_files_format_and_content: DESeq2_Output, .xlsx format - Microsoft Excel spreadsheet version 2016. Spreadsheet contains five tabs. The first three tabs are differential expression output from the DEseq2 package of strain JWCB005delta_rex strain vs. JWCB005 strain for early, mid, and late log phase, respectively. The fourth and fifth tab contain DEseq2 output differential expression analysis for late vs. early phase for strain JWCB005 and strain JWCB005delta_rex, respectively. Also included are DESeq2_input with count data and DESeq2_input_column_data with sample index information.

Submission date

Jul 30, 2017

Last update date

May 15, 2019

Contact name

Kyle Sander

E-mail(s)

[email protected]

Organization name

University of Tennessee/Oak Ridge National Laboratory

Department

Biosciences