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Sample GSM3317901

Query DataSets for GSM3317901

Status

Public on Dec 26, 2018

Title

fresh-to-saltwater 24hr control, rep 4

Sample type

SRA

Source name

gill

Organism

Fundulus heteroclitus

Characteristics

tissue: gill
Sex: male
hours post-transition to seawater: 24
arsenic: 0

Treatment protocol

Male killifish were exposed to 100 ug/L total arsenic, as sodium (meta)arsenite (Sigma-Aldrich, St. Louis, MO) or control for 48 hours, and then transferred to seawater containing the same levels of arsenic. Fish were euthanized at 0, 1 and 24 hours post-transition to seawater and gills dissected for RNA isolation.

Growth protocol

Killifish, Fundulus heteroclitus, were collected from Northeast Creek, Bar Harbor, ME and acclimated to fresh water at the MDI Biological Laboratory by gradually reducing the salinity to 10% seawater, followed by “soft” freshwater for 2 week intervals as described previously (Shaw 2007).

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) following manufacturer’s protocol.
Illumina TruSeq small RNA libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample_C4

Data processing

Read quality control diagnostic analyses using FastQC version 0.11.2.
Small RNA-Seq reads were adapter clipped using FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
MicroRNA were annotated using the miRMiner software (Tarver et al., 2013; Wheeler et al., 2009) and the killifish genome assembly (Reid et al., 2017). Using miRMiner, the clipped and length-filtered reads from all samples were collapsed into unique sequence tags. Sequence tags were aligned to miRBase stem-loop precursors in miRBase (version 21) (Kozomara and Griffiths-Jones, 2014) using NCBI BLAST (Altschul et al., 1990) to identify tags with high similarity to previously annotated miRNAs . Next, sequence tags were aligned to the genome assembly using BLAT (Kent, 2002). Alignment coordinates were analyzed to generate multiple sequence alignments of the mature miRNA products along genomic segments predicted to form stable stem-loop secondary structures by RNAFold from within the ViennaRNA Package version 1.7 (Lorenz et al., 2011). These multiple sequence alignments were examined to annotate miRNA where at least one sequence tag mapped to both the mature and star mature arms consistent with miRNA in other animals (Fromm et al, 2016). Each annotated miRNA was assigned a stem-loop precursor sequence, mature and star sequences according to the evidence in the multiple sequence alignments. miRNA homologs were identified in zebrafish (Danio rerio) miRNA families compared with eight other vertebrates in miRGeneDB (http://mirgenedb.org/) (Fromm et al., 2015)
Following miRNA annotation, the expression level of miRNAs, expressed as read counts, in each sample was calculated by aligning clipped and length filtered sequences to the annotated stem-loop precursor sequences using CLCBio Genomics Workbench version 9.0 (QIAGEN Bioinformatics, Aarhus, Denmark).
Genome_build: GCA_000826765.1 Fundulus_heteroclitus-3.0.2
Supplementary_files_format_and_content: Tab-delimited text files with mapped read counts for microRNAs. A FASTA file with miRNA sequences is included.

Submission date

Aug 02, 2018

Last update date

Dec 26, 2018

Contact name

Benjamin L King

E-mail(s)

[email protected]

Phone

207-581-2803

Organization name

University of Maine