|
| Status |
Public on Nov 01, 2020 |
| Title |
MSR-3 (Clostridium thermocellum 1313 ∆hpt∆SO3R: sulfate) |
| Sample type |
SRA |
| |
|
| Source name |
Clostridium thermocellum_1313_ culture
|
| Organism |
Acetivibrio thermocellus DSM 1313 |
| Characteristics |
genotype/variation: {delta}hpt{delta}SO3R
sulfur source: sulfate
strain: sulfite reductase knockout strain
|
| Treatment protocol |
Samples for RNA-seq were taken from mid-exponential phase (OD ~ 0.6) batch cultures grown in MTC on 5 g/l cellobiose. Control strain (∆hpt) and sulfite-reductase-mutant strain (∆hpt∆SO3R) were grown in the presence of either 5.7mM cysteine or 28.2mM sodium sulfate as the only sulfur source
|
| Growth protocol |
Strains were grown to mid-exponential phase (OD ~ 0.6) in sealed, N2 sparged 162 ml serum bottles containing 50 ml MTC (initial pH of 7.1) supplemented with 5 g/l cellobiose
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pelleted cells were resuspended in TRIzol (Invitrogen, CA), throughly mixed and then refrozen. The frozen 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 (on column) 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 Bacteria (Epicentre/Illumina). The sample was then concentrated with RNA Clean & Concentrator-5 (Zymo Research, CA) following the manufacturer’s protocol.
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.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Demultiplexing and file conversion performed by DRAGEN, version 01.003.043.02.01.02.25767
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.
Unique gene counts from the CLCBio mapping (DESeq_input.txt) were used for DESeq2 analysis to normalize and determine significant gene expression differences (DeSeq_output_all_pairwise_comparisons.xlsx)
Genome_build: NC_017304.1
Supplementary_files_format_and_content: Tab deliminated file includes unique read counts for each gene across the 4 conditions (with 3 biological replicates each) used in this study
|
| |
|
| Submission date |
Jun 14, 2017 |
| Last update date |
Nov 01, 2020 |
| Contact name |
Dawn Marie Klingeman |
| E-mail(s) |
[email protected]
|
| Phone |
+18655763435
|
| Organization name |
Oak Ridge National Lab
|
| Department |
Biosciences Division
|
| Lab |
RNA Profiling
|
| Street address |
1 Bethel Valley Rd building 15056 Rm 366 MS 6038
|
| City |
Oak Ridge |
| State/province |
TN |
| ZIP/Postal code |
37831-6342 |
| Country |
USA |
| |
|
| Platform ID |
GPL20122 |
| Series (1) |
| GSE100047 |
Sulfate assimilation in Clostridium thermocellum |
|
| Relations |
| BioSample |
SAMN07237995 |
| SRA |
SRX2919069 |
