|
| Status |
Public on Jan 31, 2017 |
| Title |
AG553 (unevolved Quad) No formate-2 |
| Sample type |
SRA |
| |
|
| Source name |
Q0 NO formate
|
| Organism |
Acetivibrio thermocellus DSM 1313 |
| Characteristics |
strain: AG553
treatment: No formate
|
| Treatment protocol |
samples were grown with and without 2 mM formate
|
| Growth protocol |
strains of Clostridium thermocellum 1313 grown in MTC5 defined media with 5 g/L cellobiose as the carbon source. Del-hpt (parental strain), AG553 (unevolved Quad) and AG601 (“best” evolved strain)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells pelleted from a 50 mL sample resuspended in 1 mL of 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 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™ 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 eluted with 20 µl of Buffer EB provided in the Min-Elute PCR purification kit (Qiagen) according to the ScriptSeq™ RNA-Seq Library preparation kit protocol. Twelve PCR cycles were used during library amplification and samples were purified using Agencourt AmPureXP magnetic beads (Beckman Coulter, IN ) 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 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 |
| |
|
| Description |
Clostridium thermocellum_1313 culture
Ct 22
|
| 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 normalize and determine significant gene expression differences
Genome_build: ASM18492v1
Supplementary_files_format_and_content: Tab deliminated file includes read counts for each gene across the 24 ( 6 conditions with 4 reps each condition) samples used in this study
|
| |
|
| Submission date |
Feb 24, 2016 |
| Last update date |
May 15, 2019 |
| 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) |
| GSE78287 |
Improved growth of Clostridium thermocellum AG553 via medium supplementation with formate and strain evolution |
|
| Relations |
| BioSample |
SAMN04515315 |
| SRA |
SRX1599913 |
