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Sample GSM2323833

Query DataSets for GSM2323833

Status

Public on Mar 07, 2017

Title

tcdd3_20dpf

Sample type

SRA

Source name

axial tissue (including bone, fat, muscle, dermis)

Organism

Oryzias latipes

Characteristics

strain: Orange-red Hd-dR
age: 20 days post fertilization
tissue: axial tissue (including bone, fat, muscle, dermis)
chemical: 0.3 nM TCDD

Treatment protocol

Medaka embryos were treated with DMSO or 0.3 nM TCDD at Stages 8-9 (approximately 4 hours post fertilization) for 1 hour. Embryos were subsequently washed four times with 1X ERM and transferred to a

Growth protocol

Following DMSO or TCDD exposure, embryos were cultured in 1X ERM at 26-27 °C with static media renewal every other day until hatch. Hatched individuals were transferred to plastic containers containing 100 mL of 1X ERM, were fed 1-2 mg of ground Otohime B1 larval diet (Reed Mariculture, Campbell, CA) daily, and media was renewed every other day until tissue dissection and RNA isolation at 20 dpf.

Extracted molecule

polyA RNA

Extraction protocol

Larval medaka were euthanized at 20 dpf in 0.125% tricaine/ERM solution and replicates were pooled to ensure 15-25 individuals per RNA sample. Specimen were transferred to Leibowitz’s media (L-15) containing 10% Fetal Bovine Serum. Dechorionated embryos or hatched larvae were passed through a 20-22 gauge hypodermic needle and 3-ml syringe several times to isolate the axial skeleton from craniofacial and abdominal viscera. Sheared tissue was collected on 105-μm nylon mesh and transferred to fresh L-15/10% FBS media. Under a dissecting light microscope axial tissue was inspected and trimmed of any remaining craniofacial or abdominal tissue. Samples were immediately flash frozen in liquid nitrogen. Tissues were homogenized in TRI Reagent® and total RNA was isolated according to the TRI Reagent manufacturer’s protocol. Finally, total RNA was quantified using Agilent 2100 Bioanalyzer and 2100 Expert Software package (Agilent Technologies). RNA samples with RNA Integrity numbers (RINs) of 9.9 or greater were used for RNA-Seq library generation.
Whole transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2 (Ion Torrent, Life Technologies, Carlsbad, CA) for the Ion PGM sequencing platform as described by the manufacturer. Briefly, polyA mRNA was purified for each sample from 2 μg of denatured total RNA with oligo(dT)25 Dynabeads®. Adaptors from Ion Adaptor Mix v2 were ligated to poly(A) RNA, and each sample was reverse transcribed to form cDNA, and then amplified using Platinum® PCR Supermix High Fidelity, Ion Xpress™ RNA 3’ Barcode Primers, and a different Ion Xpress® RNA-Seq BC Primer (BC#11-16) for each sample to form a barcoded library. The library was then pooled with equimolar amounts from each of the 6 barcoded samples. The pooled library was amplified of the pooled library using the Ion OneTouch™ 200 and Ion PGM Template OT2 200 Kit. The solution containing template-positive ISPs was enriched on the Ion OneTouch™ ES according to the manufacturer’s protocol. Sequencing primers were annealed to the template-positive ISPs, Ion PGM™ Sequencing 200 v2 Polymerase was added to the ISP solution, and incubated at room temperature for 5 minutes. Finally, the template-positive ISPs + Sequencing Polymerase mixture were carefully loaded into the Ion 318™ chip and sequenced overnight on the Ion PGM™ System.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent PGM

Data processing

The resulting reads were trimmed to remove adaptor sequences and low quality base-calls, and were aligned to the Oryzias latipes (Medaka) genome (Version 75; www.ensembl.org) using Bowtie 2.2.6.
Differentially expressed genes were identified using Cuffdiff2, whereas principal component analysis was performed with DESeq2 in R.
Genome_build: Ensembl, Version 75 ftp://ftp.ensembl.org/pub/release-75/gtf/oryzias_latipes/
Supplementary_files_format_and_content: Tab delimited text files for each sample were generated reporting FPKMs for each sampleand log2 fold change between DMSO- and TCDD-treated cohorts.

Submission date

Sep 21, 2016

Last update date

May 15, 2019

Contact name

AtLee Watson

E-mail(s)

atwatson@ncsu.edu

Organization name

North Carolina State University

Street address

Campus Box 7633