GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3070847

Query DataSets for GSM3070847

Status

Public on Apr 30, 2018

Title

350nm-400nm_2

Sample type

SRA

Source name

350nm-400nm

Organism

Xiphophorus maculatus

Characteristics

gender: male
age: 10-11 month
strain: Jp 163 B
light treatment: 350nm-400nm
tissue: skin

Treatment protocol

FL exposure occurred in a specially designed wooden box (77 cm in length, 41 cm in height, and 36 cm in depth), with a hinged wooden lid capable of sealing the interior of the box from external light. On the bottom of each of the two sides (41 cm x 36 cm) were 15.5 cm diameter high-speed fans that maintained interior temperatures of the closed box at less than 24°C. For FL exposure single fish were placed into UV transparent (UVT) plastic cuvettes (9 cm x 7.5 cm x 1.5 cm) in about 95 ml water and the cuvettes were suspended in a rack centered between and about 10 cm from the a bank of four FL bulbs (each side) inside the exposure chamber. FL sources were ‘‘cool white’’ Philips F 20T12/CW 20W, Alto (i.e., 4100K FL)) fluorescent lamps provided doses of 35 kJ/m2, that equated to an exposure time of about 40 min. For waveband exposure of X. maculatus to 50 nm [300-350 (7.3 kJ/m2), 350-400 18.6 kJ/m2), 400-450 (21.6 kJ/m2), 450-500 (21.2 kJ/m2), 500-550 (17.4 kJ/m2), and 550-600 nm (13.8 kJ/m2)] or 10 nm wavebands of light [500-510nm, 510-520, 520-530, 530-540 ,and 540-550), we utilized a TLS-300X Series Tunable Light Source (Newport Corporation, Irvine, CA, USA) containing an Ushio 300 W Xenon Short Arc Lamp Model 6258. Exposures were as detailed previously. Briefly, light emitted from the source was passed through a Cornerstone 130 Monochromator (Newport Corporation, Irvine, CA, USA) to define specific wavelengths. The bulb was burned in 15 min prior to all exposure treatments. The specific wavelengths were divided by 2 fiber optic light cables, allowing the fish to be exposed on both sides simultaneously to the defined wavelengths of light. Spectral distributions were made to determine the power output of each light source at specific wavelengths using a Newport 1918-R power meter (Newport Corporation, Irvine, CA, USA). The spectral distribution of the xenon light source was measured at full spectrum (0 nm) using an Ocean Optics STS 350-800 nm Microspectrometer (Ocean Optics Inc., Dundedin, FL, USA) and OceanView software v1.5 (http://oceanoptics.com/product/oceanview/). The microspectrometer was calibrated to a known standard using Ocean Optics Halogen Calibrated Light Source HL-3P-CAL (Ocean Optics Inc., Dundedin, FL, USA). To cover each wavelength in each 50 nm regions, the monochromator was set to scan and repeat (i.e. loop) using Asoftech Automation (http://www.asoftech.com/) through the wavelengths of each region (1 nm/sec for 50 sec) for the duration of the light exposure. Each fish was then placed in a 4 cm length x 1 cm wide x 4.5 cm height quartz cuvette filled with 14 mL of filtered aquaria water. The cuvette was then centered between the 2 fiber optic light cables and covered by a cardboard box to eliminate ambient light. After exposure, the fish was removed from the cuvette, rinsed with filtered aquaria water, placed back into a 125 mL flask filled with 100 mL of filtered aquaria water, and in the dark for 6 hours to allow for gene expression prior to sacrifice and tissue dissection.

Growth protocol

Fish utilized were mature male X. maculatus Jp 163 B, 10 to 11 months of age, from the 105th generation of sibling inbreeding were used. All fish were housed in the Xiphophorus Genetic Stock Center following standard animal mainteince protocol.

Extracted molecule

total RNA

Extraction protocol

RNA isolation was performed following the Qiagen RNeasy RNA isolation protocol (Qiagen, Valencia, CA, USA). Skin samples harvested from fish were first homogenized using a hand-held homogenizer in a 1.5 mL microcentrifuge tube while the sample remained frozen in TRI Reagent (Sigma Inc., St Louis, MO, USA). After homogenization, 300 μL of fresh 4°C TRI Reagent was added to the samples followed by incubation (rt) for 5 min. Chloroform extraction was performed by adding 120 μL chloroform and shaken for 15 sec. Samples were centrifuged (16,100 rcf for 5 min at 4°C) for phase partition. The aqueous layer was transferred to a new 1.5 mL microcentrifuge tube and a second chloroform extraction performed (300 μL TRI Reagent, 60 μL chloroform). After extraction, nucleic acids in the aqueous phase were precipitated with 500 μL 70% EtOH in diethylpyrocarbonate (DEPC) treated water. The sample was then transferred to a Qiagen RNeasy mini spin column and on-column DNase treatment was performed for 15 min at 25°C. RNA samples were then washed and eluted in 100 μL RNase free water. RNA concentration was measured with a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA). To further assess the RNA quality, a RNA integrity (RIN) score was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples processed for RNA sequencing had a RIN score above 8.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

350-400_2

Data processing

Illumina Hi-Seq 2000 was used to sequence all samples
raw reads were trimmed and filtered using a custom Perl script,The reads were truncated based on similarity to library adaptor sequences. Then, low-scoring sections of each read were removed, preserving the longest remaining fragment
Filtered reads are mapped to the Xiphophorus transcriptome using GSNAP
Gene expression were quantified using eXpress.
Genome_build: Xiphophorus maculatus Ensembl 80
Supplementary_files_format_and_content: Format: .csv; Content: gene expression profiles

Submission date

Mar 28, 2018

Last update date

Apr 30, 2018

Contact name

Yuan Lu

Organization name

Xiphophorus Genetic Stock Center

Street address

university drive

City

San Marcos