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Sample GSM2786686

Query DataSets for GSM2786686

Status

Public on Dec 11, 2018

Title

Bham2_REF(S4)

Sample type

SRA

Source name

whole organism

Organism

Daphnia magna

Characteristics

strain: Bham2
clone: Clone 2
gender: female
age: 14 days
exposure: control
culturing media: HH COMBO
replicate: rep0
molecule subtype: Genomic DNA

Treatment protocol

Maintained under standard condition with normal levels of oxygen (6 mg/L) for 14 days
The exposure design followed the OECD guidelines for assessment of chronic toxicity with some modifications (OECD, 2012). Less than 24 hours old neonates were randomly assigned to either control or exposure groups. Control Daphnia were maintained under normal laboratory conditions (oxygen concentration: 6 mgL-1). Treatments consisted of 5 days of exposure to 5-azacytidine (3.7mgL-1) or 14 days of exposure to either arsenic (100 µg L-1), hypoxia (2 mg L-1), or hyperoxia (8 mgL-1). Hypoxic and hyperoxic conditions were generated by continuous aeration of the media with 20% and 4% oxygen balanced with nitrogen, respectively. Oxygen concentrations were continuously monitored using an oxygen sensor (Unisense microrespiration system, Denmark).

Growth protocol

Daphnia magna Bham 2 clones were maintained in a 16:8 h light:dark photoperiod and temperature of 20 ± 1 °C, in high hardness COMBO medium (HH COMBO). Media were prepared using a protocol adapted from Baer & Goulden (1998) and Kilham et al. (1998), and renewed once a week. Animals were fed every other day with Chlorella vulgaris at a concentration of ≈27,550 cells of algae per individual Daphnia. Daphnia pulex EB45 and 31 clones were maintained in a 16:8 h light:dark photoperiod and temperature of 20 ± 1 °C, in standard COMBO medium (COMBO). Media were prepared using a protocol adapted from Kilham et al. (1998), and renewed every other day. Animals were fed every other day with Chlorella vulgaris at a concentration of ≈27,550 cells of algae per individual Daphnia.

Extracted molecule

genomic DNA

Extraction protocol

Daphnia samples were flash frozen in liquid nitrogen and stored in -80C until further use. DNA was extracted from the samples using MasterPure DNA purification kit (Epicentre, USA) according to the method described in our previous publication (Gonçalves Athanasio et al, 2016).
Libraries for both sodium bisulfite treated samples and input DNA samples were prepared following the EpiGnome Methyl-Seq kit (Epicentre, USA) protocol. Briefly, DNA samples (50 ng for sodium bisulfite converted and 20 ng for non-converted samples) were diluted in 9 μL of nuclease-free water, mixed with 2 μL of DNA synthesis primer and incubated at 95°C for 5 minutes. Samples were placed on ice and 5 μL of the mastermix (containing 4 μL of EpiGnome DNA synthesis premix, 0.5 μL of 100 mM DTT and 0.5 μL of EpiGnome polymerase) was added to each sample. The reactions were incubated as 25°C for 5 minutes, 42°C for 30 minutes and 37°C for 2 minutes. Then, 1 μL of exonuclease I was added to each sample and incubated for 10 minutes at 37°C, 3 minutes at 95°C followed by 2 minutes at 25°C. DNA samples were tagged by addition of TT master mix (7.5 μL of EpiGnome terminal tagging premix and 0.5 μL of DNA polymerase). Samples were incubated at 25°C for 30 minutes followed by 3 minutes at 95°C. Reactions were cooled to 4°C and purified using AMPure XP system (1.6x beads) (Beckman Coulter Inc., USA) as recommended. The final step of library construction comprised the amplification of the libraries and addition of barcodes. Each reaction contained 22.5 μL of the purified tagged DNA, 25 μL of FailSafe PCR premix E, 1 μL EpiGnome forward primer, 1 μL of EpiGnome index PCR primer and 0.5 μL of FailSafe PCR enzyme (1.25 U). PCR was performed according to the manufactures guidelines. Amplified samples were purified using AMPure XP beads (1x beads). Samples were re-suspended in 20 μL of nuclease-free water and quantity and quality of the sequencing libraries were assessed. Daphnia magna and Daphnia pulex libraries were sequenced using Illumina HiSeq-2500 platform at the Environmental Omics Sequencing Facility, University of Birmingham, UK and NextSeq platform at the Centre for Genomics and Bioinformatics, Indiana University, USA, respectively.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina NextSeq 500

Description

processed data file: cpg.var.bham2.v2.vcf.gz
processed data file: magna_snp_cgvIN.vcf.gz

Data processing

Illumina adapters (using core sequence: AGATCGGAAGAGC) and nucleotides with low quality (Phred score < 20) were removed with cutadapt (v.1.11) while processing both read pairs at the same time.
The filtered read were mapped to the reference genomes of Daphnia magna (daphmag2.4,GCA_001632505.1) and Daphnia pulex (PA42 v3.0,GCA_900092285.1) using bwa meth (v.0.10) for the bisulfite-treated samples and bwa mem (v.0.7.15-r1140) for non-bisulfite-treated samples, with default settings.
Genomic variants were called genome wide using the non-bisulfite-treated samples, with samtools mpileup (0.1.19), outputting per sample read depth, with minimum mapQ score set to 10 and without discarding anomalous read pairs. Single nucleotide polymorphism (SNP) genotypes per sample were called with bcftools (0.1.19), by excluding indels and sites were the reference was not A/C/T/G.
Single nucleotide polymorphism (SNP) at the CpG sites were called using the non-bisulfite-treated samples, with samtools mpileup (0.1.19). CpG's with observed SNP’s (with quality>50) were excluded from the methylation analysis.
Methylated CpG were called from mapped reads using MethylDackel (v0.2.1). Both singletons and discordant reads were retained, while reads with low mapping quality (<10) and nucleotides with low base calling quality (Phred<30) were excluded. Seven base pairs from the ends of reads were also excluded, as they showed an excessive amount of methylation.
Differential methylation analysis was done using methylKit (v.1.3.0).
Genome_build: GCA_001632505.1, GCA_900092285.1
Supplementary_files_format_and_content: bed (methylation counts at CpGs), vcf (SNP variation calls at CpGs), vcf (SNP variant calls genome wide)
Supplementary_files_format_and_content: txt (differential methylation analysis results: daphnia.magna.bham2.txt, daphnia.pulex.eb31.eb45.txt)

Submission date

Sep 18, 2017

Last update date

May 15, 2019

Contact name

Jouni Antero Kvist

Organization name

University of Helsinki

Department

Research Program for Molecular Neurology

Lab

Medical Neurogenetics group