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| Status |
Public on Mar 05, 2018 |
| Title |
Fermentor N°2 Sample 11h (E4) |
| Sample type |
SRA |
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| Source name |
T11h-F2/E4
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| Organism |
Clostridium beijerinckii |
| Characteristics |
strain: DSM 6423
fermentor: no. 2
time: T11h
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| Growth protocol |
Actively growing culture was inoculated at 5% into a fermentation medium (GAPES). In order to get 3 biological replicates, the same procedure was repeated 3 times on 3 different weeks. Each week, a fresh preculture was used to inoculate 2 identical bioreactors. Samples were taken over the early exponential, late exponential and stationary phases (samples at 3, 6, 8, 11, and 24h). Following centrifugation of the samples, cell pellets were immediately frozen in liquid nitrogen and supernatants were used for HPLC analysis. For each timepoint, the RNA samples of the 2 bioreactors were pooled, leading to 1 RNA sample per week and timepoint.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell lysis and RNA isolation was done with the TRIzol Plus RNA purification kit (AMbion). 5 mL TRIzol reagent is added to the frozen pellet followed by 1 mL chloroform after cell lysis. After shacking and centrifugation, the upper phase is mixed with an equal volume of 70% ethanol before transfer to PureLink RNA Spin Cartridges furnished with the kit. Kit instructions allow to obtain purified total RNA which is further processed by cleanup with the RNeasy mini kit (Qiagen). DNA is eliminated after DNAse I treatment (AM1906, Invitrogen). 20µL RNA is treated with 2 µL DNAse in 50 µL nuclease free water for 30 min at 37°C before addition of another 1.5 µL DNAse and 30 min extra incubation. Treatment is done twice and is followed by RNA cleanup with the RNeasy kit. A full quality assessment of the RNA, quantification using Nanodrop, PCR to check for residual DNA and migration on 1% agarose gel is done before depletion of ribosomal RNA with MicrobExpress kit (ThermoFischer). Depletion uses 2-10 µg total RNA in 15 µL water.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
barcode: GATCAG
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| Data processing |
In a first step, the RNA-Seq data quality was assessed by including option like reads trimming or merging/split paired-end reads. In a second step, reads were mapped onto the C. beijerinckii DSM6423 genome sequence using the SSAHA2 package that combines the SSAHA searching algorithm (sequence information is encoded in a perfect hash function) aiming at identifying regions of high similarity, and the cross-match sequence alignment program which aligns these regions using a banded Smith-Waterman-Gotoh algorithm. An alignment score equal to at least half of the read is required for a hit to be retained. To lower false positives discovery rate, the SAMtools (v.0.1.8, [22]) are then used to extract reliable alignments from SAM formatted files.
The number of reads matching each genomic object harbored by the reference genome is subsequently computed with the Bioconductor GenomicFeatures package. If reads matching several genomic objects, the count number is weighted in order to keep the same total number of reads.
Finally, the Bioconductor-DESeq package with default parameters is used to analyze raw counts data and test for differential expression between conditions
Genome_build: chromosome (circular DNA) LN908213.1 GI: 1188447636; bacteriophage (linear DNA) LN908215.1 GI: 1188453697; plasmid (circular DNA) LN908214.1 GI: 1188453683
Supplementary_files_format_and_content: The csv files report abundance measurements. Column header names: Label (Feature ID), Type (features include CDS, tRNA, rRNA, misc_RNA, fCDS), Name, Product (Putative Function), Begin (start location of feature), End (end location of feature), Frame (frame of feature), sense (Raw Count), antisense (Raw Count)
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| Submission date |
Jun 14, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Frederique Bidard |
| E-mail(s) |
frederique.bidard-michelot@ifpen.fr
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| Organization name |
IFPEN
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| Department |
Biotechnology
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| Street address |
1-4 avenue de Bois Préau
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| City |
Rueil Malmaison |
| ZIP/Postal code |
92852 |
| Country |
France |
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| Platform ID |
GPL23579 |
| Series (1) |
| GSE100024 |
RNA-seq analysis of glucose fermentation by the natural Isopropanol producer Clostridium beijerinckii DSM6423 |
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| Relations |
| BioSample |
SAMN07236922 |
| SRA |
SRX2918114 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2668041_MicroScope_data_E4.csv.gz |
164.0 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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