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| Status |
Public on Oct 26, 2014 |
| Title |
DSM 1237 stationary replicate 1 |
| Sample type |
SRA |
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| Source name |
DSM 1237_Stationary
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| Organism |
Acetivibrio thermocellus |
| Characteristics |
strain: DSM 1237
growth phase: Stationary
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| Growth protocol |
The type strains of Clostridium thermocellum, DSM 1237, 2360, and 4150, obtained from the German Type Culture collection, were employed for all growth experiments. Fresh cultures were maintained by routinely transferring 10% (v/v) mid-exponential phase inoculum into complex 1191 medium containing 2.2 g L-1 (11.8 mM) cellobiose. Cultures were grown at 60C and stored anaerobically at 4C. Cells for RNAseq sampling were grown in anaerobic Balch tubes (26 mL; Bellco Glass Inc., Vineland, NJ) in 10 mL of 1191 medium (pH 7.2) on 2.2 g L-1 cellobiose. 1191 media preparation and inoculation protocols were followed as described by Islam, Cisek, Sparling &Levin (2006). Samples were taken in exponential and stationary phase (OD600 ~ 0.37 and ~0.80, respectively).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells for RNAseq sampling were grown in anaerobic Balch tubes (26 mL; Bellco Glass Inc., Vineland, NJ) in 10 mL of 1191 medium (pH 7.2) on 2.2 g L-1 cellobiose. 1191 media preparation and inoculation protocols were followed as described by Islam et al. Samples were taken in exponential and stationary phase (OD600 ~ 0.37 and ~0.80, respectively). Cell pellets were stored at -80 °C in 1 ml RNAlater until ready for processing. Total RNA was extracted from 10 ml mid-exponential and late exponential (OD600 ~ 0.37 and ~0.80, respectively) Clostridium thermocellum cultures using the RNeasy Mini kit (QIAGEN, Valencia, CA, USA) protocol as described for gram-positive bacteria, but with the following modifications. Cell pellets were collected by centrifugation and re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 20 mg/ml lysozyme and incubated at 37 °C for 30 minutes. To ensure efficient lysis, a mechanical shearing step was included that involved passing the cell pellets 10 times through a 22G syringe prior to the addition of ethanol. Total RNA was eluted in 30 μl RNase-free water. Following the first elution, the entire volume was re-applied to the mini column, incubated for 3 minutes and finally centrifuged at 8000 x g for 1 minute.
Illumina Truseq mRNA
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
D14
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| Data processing |
Basecalls performed using CASAVA version 1.8
Trimming step – Adaper clipping and quality filtering: Raw illumina reads were trimmed and filtered by Trimmomatic-0.22 with parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 and all the occurred adapters in the first 8k paired end raw reads from "Illumina Adapter Sequences Letter" for clipping accompanying parameters <seed mismatches>:<palindrome clip threshold>:<simple clip threshold> - 2:40:15.
FPKM (fragments per kilobase of exon per million fragments mapped) files were generated with Bowtie2-2.0.2, Tophat-2.0.6, Cufflinks-2.0.2 with commands 1. tophat2 -p 24 -r 300 --mate-std-dev 50 -o $Tophat_PairedOUTPUT -G $GTF $BINDEX $R1,$R2; 2. bed_to_juncs < $Tophat_PairedOUTPUT/junctions.bed > $Tophat_PairedOUTPUT/unpaired_list.juncs; 3. tophat2 -j $Tophat_PairedOUTPUT/unpaired_list.juncs -p 24 -r 300 --mate-std-dev 50 -o $Tophat_UnpairedOUTPUT -G $GTF $BINDEX $Unpaired; 4. samtools sort $bam_file1 $merge_bam/paired.sort; 5. samtools sort $bam_file2 $merge_bam/unpaired.sort; 6. samtools merge -f $merge_bam/total.bam $merge_bam/paired.sort.bam $merge_bam/unpaired.sort.bam; 7. cufflinks --max-bundle-frags 4000000 -b $TRANSINDEX -u -o $Cufflinks_OUTPUT -G $GTF -p 12 -v $merge_bam/total.bam
Supplementary_files_format_and_content: Paired and unpaired fastq files from trimming step – each sample has two files of paired reads R1 and R2 and a file of unpaired reads
Supplementary_files_format_and_content: tab-delimited text files include FPKM values from Cufflinks for each Sample
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| Submission date |
Oct 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Sparling |
| E-mail(s) |
Richard.Sparling@umanitoba.ca
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| Organization name |
University of Manitoba
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| Department |
Microbiology
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| Street address |
418 Buller Building
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| City |
Winnipeg |
| State/province |
MB |
| ZIP/Postal code |
R3T 0M2 |
| Country |
Canada |
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| Platform ID |
GPL17844 |
| Series (2) |
| GSE51744 |
University of Manitoba/MGCB2 C thermocellum RNA-seq Experiment |
| GSE51745 |
Comparison of gene expression profiles in three Clostridium thermocellum strains: DSM 1237, 2360 and 4150 |
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| Relations |
| BioSample |
SAMN02384675 |
| SRA |
SRX368290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1251937_D14_genes.fpkm_tracking.gz |
127.3 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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