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Sample GSM3230585

Query DataSets for GSM3230585

Status

Public on Jun 18, 2019

Title

E41 high TBPH exposure

Sample type

SRA

Source name

embryo

Organism

Fundulus heteroclitus

Characteristics

tissue: embryos
treatment: high dose of TBPH solution

Treatment protocol

Each ELA used embryos exposed individually from one to seven days post-fertilization (dpf) in ten mL of amended sea water. Following these early developmental exposures, embryos were flash frozen by immersion in liquid nitrogen and archived at -80° C until genomic or chemical analysis or allowed to develop until seven days post hatching (dph)

Growth protocol

Adult killifish (~100 - 200 fish) were collected using baited traps at an uncontaminated estuarine site in Barnstable, MA (‘Scorton Creek’; Nacci et al. 2010). Fish were returned to US EPA ORD aquarium facilities (Narragansett, RI), and maintained in ~300 – ~600 L flowing sea water tanks for > six months or up to two years, and then used as breeding stock. These stocks provided hundreds of embryos that were collected following unstimulated or manually induced spawning events, held at 23° C overnight, then screened to remove dead or abnormal embryos. Typically, batches of normally developed embryos from a single tank of breeding stock were used in a single ELA

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from embryos using the MagMAX™-96 Total RNA Isolation kit (ThermoFisher, Waltham, MA) following the manufacturer’s protocol supplied with the kit, except as noted below. Homogenization of embryos was performed in 1.5 ml microcentrifuge tubes using a Bullet Blender© Storm 24 homogenizer (Next Advance, Troy, NY) with a 3.2 mm stainless steel bead added to each sample. Each embryo was homogenized in 100 µl of MagMax Lysis/Binding solution supplemented with 0.7 µl β-mercaptoethanol. Contaminating DNA was removed from samples using the supplied Turbo DNase. Both the duration of DNase treatment and incubation temperature were increased, to 20 min and 37 °C, to increase the effectiveness of digestion. RNA was eluted from the magnetic beads using 25 µl of Elution Buffer. During the elution step, samples were incubated at 37 °C
Libraries were prepared (n = 16 per treatment) using the TruSeq Stranded mRNA Library Prep Kit for Neoprep (Illumina, San Diego, CA) according to the manufacturer’s protocol. One hundred ng RNA was used as input into the automated NeoPrep System (Illumina, San Diego, CA). The NeoPrep is able to prepare libraries for up to 16 samples. Samples were blocked by treatment for a multiplex level of 16 samples. This level of multiplexing provides a theoretical sequencing depth of 18.75 M sequences per sample. Following library preparation, individual samples were quantified using the Qubit fluorometer (Thermofisher), normalized to 10 nM and pooled

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

TBPH_FPKM_dat.txt

Data processing

base calling was done by Illumina Real Time Analysis (RTA) v2.7.7
read quality was checked for each sample using FastQC
raw reads were mapped to the transcript sequences of Fundulus heteroclitus’ reference genome from NCBI using BWA default paired-end read mapping protocol
the reads that were not mapped in-pair were filtered out
total read count of each transcript was reported and normalized by EpiCenter
RPKM data were derived from the normalized read counts
Genome_build: version 3.02
Supplementary_files_format_and_content: RPKM data in the tab-delimited text format

Submission date

Jun 28, 2018

Last update date

Jun 18, 2019

Contact name

Weichun Huang

Organization name

US EPA

Department

ORD

Street address

109 TW Alexander Dr