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Sample GSM2865881

Query DataSets for GSM2865881

Status

Public on Nov 28, 2017

Title

JWCB005Δcra - NaCl - 3

Sample type

SRA

Source name

Caldicellulosiruptor bescii strain JWCB005 with the cra gene deleted cell biomass

Organism

Caldicellulosiruptor bescii

Characteristics

strain: Caldicellulosiruptor bescii strain JWCB005 with the cra gene deleted
osmolyte added to media: Sodium Chloride
genotype: delta-pyrF delta-cra
total media starting osmolarity: 300 mOsm/L
primary osmolyte: sodium chloride

Treatment protocol

Each single gene deletion strain was incubated separately in three elevated osmolarity conditions; added sodium chloride to a total caluclated initial osmolarity of 300 mOsm/L, added glucose to a total caluclated initial osmolarity of 500 mOsm/L, and added glucose to a total starting calculated osmolarity of 300 mOsm/L.

Growth protocol

Cells were cultured in 135 mL serum bottles in a working/culture volume of 50 mL at a growth temperature of 75oC. Media was prepared and allowed to deoxygenate overnight in an anaerobic chamber containing 5% H2, 10% CO2 and 85% N2. Appropriate amounts of various osmolytes were added to media to give each media its total calcuated initial osmolarity. Bottles were inoculated to equivalent OD680’s of 0.001-0.01 using batch grown cultures grown to mid-log phase. Cultures were incubated in a shaking incubator oscillating at 200 rpm. Three cultures were maintained and sampled for optical density measurement to track growth. Six parallel bottles were maintained and harvested when they achieved mid-log (OD680 = ~0.170-0.190). The three harvested replicates with OD680 closest to 0.180 were processed for RNAseq analysis.

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

Fastq files were downloaded from HudsonAlpha’s servers and verified for integrity by computing their checksum. Read quality was checked using FastQC (v. 0.11.5) [279]. RNAseq reads were trimmed the using Trimmomatic (v. 0.33) MAXINFO method with target length and strictness parameters set to 40 and 0.8, respectively [280]. Reads were mapped to the genome of Caldicellulosiruptor bescii JWCB005 (GCF_900166995.1, last modified 2017/03/09) using Bowtie2 (v. 2.2.9) with the same parameters as the very-sensitive preset option expect the number of mismatches was set to 1 [281], and reads were counted with HTSeq (v. 0.6.1p1) [282].
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: NZ_FUZN01000002
Supplementary_files_format_and_content: binary_comparisons_glu_gly_nacl_fapR_cra.xlsx, .xlsx format - Microsoft Excel spreadsheet version 2016. Spreadsheet contains six tabs. Each tab contains differential expression output from the DEseq2 package comparing any of the six [deletion strain-osmolyte] combinations with that of the genetic parent strain JWCB005 grown in the same osmolyte condition (e.g. JWCB005ΔB5X54_RS01260 vs. JWCB005, both strains grown in elevated glycerol). The strain comparisons and osmolyte conditions are indicated on the top of each worksheet.

Submission date

Nov 27, 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