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| Status |
Public on Jun 03, 2020 |
| Title |
CL805 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Scomber japonicus |
| Characteristics |
exposure: Control
Stage: adult
gender: male
tissue: liver
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| Treatment protocol |
For exposure experiments (for both species), five males were exposed to 12.5 pM EE2 for five hours (environmentally relevant concentration in the range of surface water levels) and an additional unexposed five males served as controls.
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| Growth protocol |
Sardine and mackerel were obtained from the live bait receiver operated by Everingham Bros Inc. in Mission Bay, San Diego, California. Fish were allowed to acclimate to research tanks at the NOAA Southwest Fisheries Science Center Experimental Aquarium facility under ambient temperature (~17oC) flow through seawater conditions and fed a pelleted feed (Bio-Oregon) to satiation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Fish were euthanized with an overdose of Tricaine Methanosulfonate (250mg/L) dissolved in seawater. Livers were harvested and flash-frozen in liquid nitrogen at -196oC. These procedures followed an approved institutional IACUC protocol and all animals were treated humanely. RNA was extracted from liver samples using TRIzol reagent (Invitrogen, CA) and further purified using the RNeasy Mini kit with DNAse to remove DNA (Qiagen, Valencia, CA). RNA concentrations were determined at 260 nm using an ND1000 (Nanodrop, Wilmington, DE). RNA was tested for structural integrity with the 6000 Nano LabChip assay from Agilent, (Santa Clara, CA, USA). Only RNA samples with RIN scores > 7.0 were used for RNA-seq.
Libraries for RNA sequencing (RNAseq) were generated using Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, USA) in accordance with the manufacturer’s recommendations (Figure 2A). The library fragments were purified with AMPure XP system (Beckman Coulter, USA) to select cDNA fragments of approximately 300 bp in length. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10-cycle PCR reaction. Products were purified using AMPure XP system and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
mackerel_control_vs_exposed.txt
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| Data processing |
Clustering was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA). After cluster generation, the libraries were sequenced on an Illumina GAIIx and 101-bp single-end reads were generated.
Data was formatted to FASTQ format using CASAVA v1.8 (Illumina). Sequenced reads (fastq files) were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
To analyze the sardine and mackerel RNAseq data, an analytical pipeline was developed that starts with the conversion of fastq files to fasta format via a custom PERL script. As comprehensive genomic sequence data for sardine and mackerel is presently not available, we exploited the phylogenetic similarity of sardine and zebrafish and mapped sardine reads to the zebrafish (Danio rerio) Genome Reference Consortium Zebrafish Build 10 (GRCz10) transcriptome. For mackerel, we mapped reads to the reference transcriptome, fugu (Takifugu rubripes) Takifugu rubripes FUGU 4.0 using the Nucleotide-Nucleotide Basic Local Alignment Search Tool (BLAST) nucleotide to nucleotide option (blastn) (Altschul et al. 1990).
After determining the threshold alignment length the reads were assembled into transcript level expression summaries; the number of reads mapped to each gene or transcript, with alignment length greater than the threshold alignment length, were summed, yielding a count as a measure of transcript expression.
In order to infer differential gene expression with robust statistical power, we utilized DEseq2. Transcript count data from DESeq2 analysis was ranked according to adjusted p-value (or q-value), the smallest false discovery rate (FDR) at which a transcript is called significant. FDR was calculated using the Benjamini-Hochberg multiple testing adjustment procedure and the cut-off was set at q ≤ 0.1.
Genome_build: GRCz10 & FUGU 4.0
Supplementary_files_format_and_content: tab-delimited .txt files include the DESEQ2 output for the Control vs EE2 exposed comparison for both fish species. Columns to the right of the Base Mean column represent DEseq2 output. baseMean is the mean value of the read counts for that gene across all samples. The log2FoldChange tells us how much the gene's expression has changed due to EE2 exposure. This value is reported on a logarithmic scale to base 2. The lfcSE value is the standard error estimate for the log2 fold change estimate. The p-value and pad-j represent uncorrected and corrected, i.e. Benjamini and Hochberg (False Discovery Rate). For the sardine data the ID is a zebrafish gene identifier, i.e. the sardine gene mapped to zebrafish NCBI Reference Sequence. The human Ortholog GeneID was determined via Ensembl homology and the corresponding HUMAN ORTHOLOG SYMBOL is provided in addition to aliases and a description of the gene. For the mackerel data the ID for the JGI Fugu ID (TAKRU4) gene is provided (first two columns), The Fugu_Ensembl_transcript, Fugu_Ensembl_protein and Fugu_Ensembl_gene information are presented. The human ortholog determined via Ensembl homology (Human gene ID), Gene Symbol, NCBI Protein ID (human), REFSEQ: accession (human) and a description of the gene are provided.
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| Submission date |
Apr 27, 2018 |
| Last update date |
Mar 26, 2021 |
| Contact name |
Gary Hardiman |
| E-mail(s) |
hardiman@musc.edu, g.hardiman@qub.ac.uk, glen@musc.edu
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| Organization name |
Medical University of South Carolina
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| Department |
Medicine
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| Street address |
135 Cannon Street
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| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
SC |
| Country |
USA |
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| Platform ID |
GPL24945 |
| Series (1) |
| GSE113780 |
Transcriptomic analysis of short-term 17α-ethynylestradiol exposure in two Californian sentinel fish species sardine (Sardinops sagax) and mackerel (Scomber japonicus) |
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| Relations |
| BioSample |
SAMN08995500 |
| SRA |
SRX4004199 |
| 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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