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| Status |
Public on Nov 30, 2016 |
| Title |
Dodecane Replicate 1 |
| Sample type |
SRA |
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| Source name |
Bacterial
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| Organism |
Acinetobacter venetianus RAG-1 = CIP 110063 |
| Characteristics |
Stage: Mid log phase cells
carbon source: dodecane
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| Treatment protocol |
A. venetianus RAG-1 was grown in presence of three different carbon sources, specifically, dodecane (1 % v/v), dodecanol (5mM) and sodium acetate (0.2 %) in triplicates, in 250 ml Erlenmeyer flasks with 50 ml of E2 media. The cells were harvested at mid-log phase, frozen in liquid nitrogen and stored at -80 °C. Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany).
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| Growth protocol |
Acinetobacter venetianus RAG-1 (ATCC 31012) was obtained from ATCC and maintained on E2 medium(23) with either 1 % v/v dodecane(13) or 0.01 % v/v ethanol(24). Our preliminary data indicated that replacement of the trace metal solution in E2 medium with ferrous chloride lead to increased growth rates and hence E2 medium was supplemented with freshly prepared ferrous chloride (0.05 mg/ml final volume).
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| Extracted molecule |
total RNA |
| Extraction protocol |
The extracted RNA was subjected to Turbo DNAse to get rid of any DNA contamination. The RNA obtained was analyzed using Agilent 2100 Bioanalyzer with the Agilent RNA 6000 Nano Kit. The RIN (RNA Integrity Number) values of all samples were above 9.6 indicating high quality RNA. The total RNA samples were prepared for Illumina Next-Generation Sequencing at the Functional Genomics Lab (FGL, QB3-Berkeley Core Research Facility at University of California, Berkeley). RiboZero kit (Illumina) was used to deplete ribosomal RNA. The treated RNA was re-checked on Bioanalyzer for integrity.
The library preparation was performed on the Apollo 324TM with PrepXTM RNA-Seq Library Preparation Kit (WaferGen Biosystems, Fremont, CA) according to the manufacturer’s recommendation. The resulting samples were quantified using fluorimetry (Qubit, ThermoFischer) and PCR amplified for 18 cycles to incorporate indexes and flow cell-binding regions.
Final libraries were quantified by Qubit, Bioanalyzer and qPCR, pooled at 14 picomolar concentration and sequenced on an Illumina HiSeq2000 using 50 base single read version 3 chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The resultant bclfiles were converted to Fastq files and demultiplexed using the Illumina CASAVA software v1.18.
The A. venetianus RAG-1 genome (NCBI accession number APPO00000000.1) was uploaded to RAST(26–28) server for annotation. The trimmed and rRNA-depleted RNA-Seq reads were mapped against the RAST-annotated A. venetianus genome using the CLC Bio Genomics Workbench 8.0.2 software (http://www. clcbio. com/products/clcgenomicsworkbench), which re-implemented EdgeR RNA quantification workflow(29).
The reads per kilobase of exon model per million mapped reads (RPKM) method was used to normalize expression of each gene(30). The differential gene expression tests were conducted with tagwise dispersion with the following parameters: length fraction cutoff=0.95, similarity fraction cutoff=0.8, exact test=”all pairs,” reads count filter=5, max P-value=0.01, and minimum fold change value=2.
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| Submission date |
Feb 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ankita Kothari |
| E-mail(s) |
[email protected]
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| Phone |
4803997899
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| Organization name |
Lawrence Berkeley National Lab
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| Lab |
Aindrila Mukhopadhyay
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| Street address |
5885 Hollis Str
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| City |
Emeryville |
| State/province |
CA |
| ZIP/Postal code |
94704 |
| Country |
USA |
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| Platform ID |
GPL21501 |
| Series (1) |
| GSE78186 |
Transcriptomic analysis of the marine oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization. |
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| Relations |
| BioSample |
SAMN04510312 |
| SRA |
SRX1597375 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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