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Sample GSM5091885

Query DataSets for GSM5091885

Status

Public on Feb 19, 2021

Title

Normoxic Control 3

Sample type

SRA

Source name

whole body; n = 5 larvae

Organism

Cyprinodon variegatus

Characteristics

age: 8 days post-hatch
oil concentration (tpah50, ug/l): 0.3
salinity (ppt): 17.0 +/- 2.0
oxygen concentration: greater than or equal to 5 mg/ml

Treatment protocol

Larvae were exposed to one of four conditions: control, hypoxia, oil, or oil plus hypoxia. Larvae (n =20) were placed in mesh cups, with three replicate cups per treatment. All larvae were exposed for 48 h and depurated in clean, normoxic water for an additional 48 h to allow stabilization of any treatment induced effects to methylation patterns. Exposures were conducted in flow through systems delivering test solutions at a rate of 2L/h. Hypoxic conditions were achieved via bubbling of nitrogen gas through test solutions until dissolved oxygen reached £ 2 mg/L O2. Environmental conditions were held at 15 ppt and 30°C across treatments and tanks were monitored daily for temperature and salinity. Dissolved oxygen was monitored every six hours to ensure maintenance of oxic regimes. Following 48h of depuration, larvae were sampled and stored in RNA later at -80°C until further analysis.

Growth protocol

C. variegatus brood stock were maintained at the University of Southern Mississippi’s Toxicology Lab in Ocean Springs, MS. Fish were kept in 300 L circulating raceways containing 15 ppt artificial sea water at 25°C and 15 ppt salinity on a 16:8 light/dark cycle and fed commercial flake food daily. Breeding was initiated by the placement of breeding nets in raceways. After 12 hours embryos were collected and examined for fertilization. Embryos were rolled on mesh mats to remove external villi, and incubated in 15 ppt sea water at 30°C with agitation to prevent clumping. After hatching, embryos were reared until yolk-sacks were absorbed and larvae began free-feeding (4 days post-hatch). Upon initiation of feeding, larvae were fed Artemia nauplii once daily. Feeding continued throughout experiments.

Extracted molecule

genomic DNA

Extraction protocol

DNA was extracted and RNAse treated using DNeasy Blood and Tissue kit.
To fragment DNA for immunoprecipitation, 8µg of DNA per sample was digested using MseI and DraI restriction. Resulting fragments were end repaired using the large (Klenow) fragment of DNA polymerase. Purified, fragmented DNA (3 µg) was diluted in 450 µL of ultra-pure water, and heat denatured at 95°C for 10 minutes. Samples were then immediately placed on ice to prevent re-annealing of fragments. Following cooling, 50 µL of 10x immunoprecipitation (IP) buffer and 4µL of 5-methyl cytosine (5meC) antibody. Samples were then incubated overnight with rotation at 4°C. For each sample, 50 µL of MagnaBind Goat Anti-Mouse IgG magnetic beads were added to each sample and incubated with rotation for 2 h at room temperature. Following incubation, beads were collected using a magnetic rack, and supernatent discarded. Beads were then washed three times in 1X IP buffer, and resuspended in in 500μL of proteinase K digestion buffer containing 5μL/mL proteinase K solution (stock concentration 20 mg/mL). Proteinase K reactions were incubated with rotation for 2 h at 50°C. After incubation, beads were collected using a magnetic rack and the supernatant collected. To purify immunoprecipitated DNA, the supernatant from each reaction was PCI purified according to published protocols.
Libraries were prepared using ACCEL-NGS® 1S Plus & Methyl-SEQ kit (Swift Biosciences, Ann Arbor, MI.) and sequenced on an Illumina NovaSeq to generate 2 x 150 bp reads.

Library strategy

MeDIP-Seq

Library source

genomic

Library selection

5-methylcytidine antibody

Instrument model

Illumina NovaSeq 6000

Description

Hypoxia_dif_meth.csv.gz; Oilhypoxia_dif_meth.csv.gz

Data processing

Bases Called using Illumina HiSeq Control Software
Initial processing of data was performed using CLC Genomics Workbench 12 (Qiagen, Hilden, Germany). Adaptor sequences and low quality reads were trimmed, paired ends matched, and reads aligned to the C. variegatus genome
Differentially methylated regions were identified using the MEDIPS R package (v 1.34).
To calculate differential methylation, the C. variegatus genome was divided into consecutive 100 bp windows, and read depth for each window compared between control and treatment samples.
The edgeR Bonferroni adjusted p-value was used to identify differential read coverage between groups; regions were considered differentially methylated at edgeR p adj. of £ 0.1
Genome_build: NCBI genome ID 13078
Supplementary_files_format_and_content: edge R output (CSV files) including TPM for each 100 bp window.

Submission date

Feb 18, 2021

Last update date

Feb 20, 2021

Contact name

Elizabeth R. Jones

E-mail(s)

[email protected]

Phone

843-661-1899

Organization name

Francis Marion University