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Sample GSM2203906

Query DataSets for GSM2203906

Status

Public on Oct 10, 2016

Title

96 hpf AB Diquat rep 3

Sample type

SRA

Source name

30 pooled zebrafish wildtype embryos (AB background), exposed to 20 μM Diquat. RNA sampled at 96 hours post-fertilization (hpf).

Organism

Danio rerio

Characteristics

developmental stage: embryo
genotype: wildtype

Treatment protocol

Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hours post fertilization (hpf), 48hpf, and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment, 20µM diquat or 1mM tBOOH for a 4-hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis, animals were moved to 0.3x Danieau’s for 48 hours post-exposure (hpe) and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA-Seq experiments, animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at -80°C.

Growth protocol

Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated, filtered, and re-circulated system water (28.5 °C) and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background (Zebrafish International Resource Center, Eugene, OR). Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee (Animal Welfare Assurance Number Number A3320-01) and the University of Michigan Institutional Animal Care and Use Committee (Animal Welfare Assurance Number Number A3114-01). To create a germline nfe2 knockout, a pair of vectors containing TAL effector nucleases (TALENs) targeting exon 3 of nfe2 were generated using the REAL (Restriction Enzyme And Ligation) assembly method. Component plasmids were obtained from Addgene (www.addgene.org/talengineering/talenkit/). Briefly, target sites were selected and TALENs designed using Zifit (http://zifit.partners.org/ZiFiT/), followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background (Shavit et al, in preparation). The TALEN target sequences are: 5'- TCACCCACCTCTTATGAG-3' and 5'- CATGACTACACGTGGTCA-3'. A subsequent founder deletion of eight base pairs (GCACATGA) was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift, causing a change in protein sequence starting at amino acid 111 (M→ D) and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar (CNC) family basic leucine zipper domain that is responsible for DNA binding.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit (BioRad, Hercules, California, USA) following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA), the quality of the RNA was checked using Caliper Instrumentation (PerkinElmer, Waltham, MA, USA) and RiboGreen reagents (Invitrogen, Carlsbad, CA, USA).
Directional mRNA libraries with poly(A) selection were made using New England Biolabs (Ipswich, MA, USA) reagents: NEBNext Poly (A) mRNA Isolation Magnetic Module, NEBNext First Strand Synthesis Module, NEBNext Second Strand Synthesis Module (with dUTP), NEBNext End Repair Module, NEBNext dA Tailing Module, and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen (Thermo Fisher, Waltham, MA, USA) and Caliper instrumentation (PerkinElmer, Waltham, MA, USA). 50 base pair (bp), paired end, libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

30 pooled zebrafish wildtype embryos (AB background), exposed to 20 μM Diquat. RNA sampled at 96 hours post-fertilization (hpf). Replicate 3

Data processing

Raw RNA-Sequencing (RNA-Seq) reads were obtained from HudsonAlpha as 50bp paired-end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality, per sequence GC content, sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool (Version 0.11.4).
Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 (Version 2.0.14), which functions based on Bowtie2 alignment software (Version 2.2.5) using a mean inner distance of 300 bp, a maximum intron length of 380,000bp and all other parameters set to default values.
Read counts for each gene were then obtained for the aligned reads using the Htseq-counts tool (Version 0.6.1). The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference, with pre-sorting by name through Samtools Sort (Samtools version 0.1.19) within the Htseq-count software. Raw counts data obtained from Htseq-counts were normalized using DESeq2 (Version 1.10.1) in R (version 3.2.3, 64bit platform) using size factors obtained from the total dataset and virtual reference sample based on the data.
Genome_build: GRCz10
Supplementary_files_format_and_content: EXCEL DeSeq2 normalized counts

Submission date

Jun 17, 2016

Last update date

May 15, 2019

Contact name

Andrew G. McArthur

E-mail(s)

[email protected]

Organization name

McMaster University

Department

Biochemistry & Biomedical Sciences