 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 23, 2021 |
| Title |
C. bescii RNA-Seq cellobiose 3 |
| Sample type |
SRA |
| |
|
| Source name |
cell pellet
|
| Organism |
Caldicellulosiruptor bescii DSM 6725 |
| Characteristics |
agent: 5 g/L cellobiose
|
| Growth protocol |
Five different growth substrates were used (glucose, cellobiose, crystalline cellulose (Avicel), xylose, birchwood xylan (Sigma)). Growth experiments were performed in triplicate at 78°C as closed cultures without pH control (400 mL volume, shaken at 150 rpm).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets collected from 200 ml samples drawn from each fermenter were resuspended in 2 mls of TRIzol (Invitrogen, CA) and lysed by sonication at 40% power (9W) on a Misonix 3000 sonicator (Farmingdale, NY). Samples were sonicated for a total of 15 seconds (3 x 5 second pulse followed by 1 minute off). Chloroform was added, and the sample was mixed and centrifuged. The aqueous layer was removed and mixed 1:1 with 80% ethanol. Then the mixture was processed on a RNeasy column (Qiagen Hilden, Germany) following the manufactures protocol and the on-column DNase digestion. RNA was eluted off the column in 35 µL RNAse free H20. The RNA was quantified using a Nanodrop 1000 instrument (ThermoScientific, Waltham, MA) and RNA quality was accessed using a Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and a Nano6000 chip kit. Purified total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal kit for Gram Positive Bacteria (Epicentre-Illumina, WI) following the manufactures protocol. The depleted sample was concentrated on a 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 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.
Epicentre (Illumina) ScriptSeq mRNA-Seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
cellobiose3
|
| Data processing |
Raw reads were first assessed for sequencing quality using SolexaQA++ tool kits.
raw reads were trimmed by using BWA dynamic trimming algorithm in the SolexaQA++ tool kits to obtain the high-quality reads (phred score > 25 and length after trimming > 25 bp).
High-quality reads were aligned to the C. bescii DSM 6725 (NC_012034) genome using the Stampy aligner software with default parameters to generate Bam files.
Raw count table was generated from the bam files using BEDTools.
Raw count table was then used for statistical analysis to identify differential gene expression using the voom method.
Supplementary_files_format_and_content: Tab-delimited text file with raw read counts for each gene and each sample.
Supplementary_files_format_and_content: Excel table with log2fold change for average Voom log2 expression values (normalized by library size) for each gene and each pair of conditions.
|
| |
|
| Submission date |
Dec 18, 2020 |
| Last update date |
Feb 23, 2021 |
| Contact name |
Michael W Adams |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
University of Georgia
|
| Department |
Department of Biochemistry and Molecular Biology
|
| Street address |
220 South Jackson Street
|
| City |
Athens |
| State/province |
Georgia |
| ZIP/Postal code |
30602 |
| Country |
USA |
| |
|
| Platform ID |
GPL29511 |
| Series (2) |
| GSE163472 |
Transcriptional regulation of plant biomass degradation and carbohydrate utilization genes in Caldicellulosiruptor bescii [RNA-Seq] |
| GSE163475 |
Transcriptional regulation of plant biomass degradation and carbohydrate utilization genes in Caldicellulosiruptor bescii |
|
| Relations |
| BioSample |
SAMN17112469 |
| SRA |
SRX9702125 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
