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| Status |
Public on Oct 19, 2017 |
| Title |
Control 4 |
| Sample type |
SRA |
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| Source name |
embryo 72 hours post fertilization
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| Organism |
Danio rerio |
| Characteristics |
treatment: no exposure
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| Treatment protocol |
Arsenic was presented to the zebrafish embryos in the form of sodium arsenite (NaAsO2; CAS 7784-46-5; Sigma-Aldrich, St. Louis, MO, USA), further referred to as arsenite. A stock solution of arsenite was prepared in and further diluted with OECD medium (294.0 mg/L CaCl2∙2H2O, 123.3 mg/L MgSO4∙7H2O, 64.7 mg/L NaHCO3, 5.7 mg/L KCl; (OECD, 2002)). The concentrations of arsenite were in the range of 0, 0.1 and 1 mM. The collected fertilized eggs were rinsed with OECD medium, distributed among the test concentrations, and embryos of matched developmental stage were then distributed over triplicate experimental units which were 20 embryos in 2 mL per embryo per well in a 24-wells plate for the range finding experiment or pools of embryos (n=20) in petri dish at 2 mL per embryo, and further kept in an incubator at 26.5 ± 1 °C with a day-night cycle of 14-10 h. The embryos were scored for morphological development and teratogenicity using a standardized scoring system (Hermsen et al., 2011), which considers 9 develomental parameters, such as development of the eyes, pectoral fins, head, and pigmentation, with a maximum score of 15 points at 72 hours post fertilization (hpf).
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| Growth protocol |
Danio rerio adults were commercially obtained wild-type import (Ruinemans Aquarium BV, Montfoort, the Netherlands) and maintained and bred for more than 8 years/generations in our lab. Adult zebrafish were kept as groups of 20-30 in 7.5L tanks in a ZebTec system with automatically controlled water parameters at 26.5 ± 1°C with a day-night cycle of 14-10h. Small spawning groups of 2m/2f, which had been separated 3 days before, were composed on the day before spawning, which was triggered by morning light on the next day. Eggs were collected within the next 2 hours and assessed for quality. Spawns with >10% coagulations or deformities, i.e. damaged membranes or embryo asymmetries, were discarded.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from the exposed pools of embryos using the Gentra Puregene Tissue kit (Qiagen, Hilden, Germany).
Genomic DNA (2.5 micrograms) was spiked with 8 methylation standards with defined methylation levels of ranging from 0 to 100%. Spiked DNA was digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. SmaI cuts only unmethylated sites leaving blunt ends. The enzyme is completely blocked by CpG methylation. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. XmaI can cut both unmethylated and CpG methylated sites (CCmeCGGG) leaving 5’CCGG overhangs. The unmethylated sites thus have the GGG signature whereas the methylated sites have the CCGGG signature at the 5’ ends of the restriction fragments. Digested DNA was supplemented with dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3'>5' exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3' DNA ends created by XmaI digestion and added 3' dA tails to all fragments. Restriction fragments with methylation-specific signatures were ligated to NEBNext Illumina adapters and amplified using NEBNext® Multiplex Oligos for Illumina®, KAPA HiFi HotStart ReadyMix PCR Kit and 12 PCR cycles.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequencing tags were mapped to SmaI sites in the Danio rerio genome (danRer7) and signatures corresponding to methylated (starting with CCGGG) and unmethylated (starting with GGG) CpG were enumerated for each SmaI/XmaI site.
At each SmaI/XmaI site, let sm be the number of tags corresponding to methylated CpG (i.e., tags starting with CCGGG) and u be the number of tags corresponding to unmethylated CpG (i.e., tags starting with GGG), the methylation value was calculated as 100%*(c*sm/(c*sm+u)), where c is the correction factor calculated based on observed and expected methylation of spiked-in standards.
Genome_build: danRer7
Supplementary_files_format_and_content: tab delimited text file with numbers of reads and percent methylation at individual CCCGGG sites
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| Submission date |
Dec 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jaroslav Jelinek |
| E-mail(s) |
jjelinek@coriell.org
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| Phone |
8567579739
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| Organization name |
Coriell Institute for Medical Research
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| Department |
Research Labs
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| Street address |
403 Haddon Avenue
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| City |
Camden |
| State/province |
NJ |
| ZIP/Postal code |
08103 |
| Country |
USA |
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| Platform ID |
GPL18413 |
| Series (1) |
| GSE90735 |
Sodium arsenite effects on DNA methylation in zebrafish embryos. |
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| Relations |
| BioSample |
SAMN06093169 |
| SRA |
SRX2388238 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2411598_ZFE_094-24066045.txt.gz |
568.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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