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| Status |
Public on Oct 18, 2019 |
| Title |
WGBS_F_EB45.2 |
| Sample type |
SRA |
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| Source name |
whole organism
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| Organism |
Daphnia pulex |
| Characteristics |
strain: Eloise Butler (45)
clone: Clone 45
gender: female
age: Mix of ages of 3, 8 and 15 days
tissue: whole body
exposure: control
culturing media: Standard COMBO
replicate: rep2
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| Treatment protocol |
To induce male Daphnia, sexually matured individual female Daphnia were treated with the crustacean reproductive hormone, methyl (2E, 6E)-farnesoate (MF) at a final concentration of 400nM. This concentration is sufficient to induce male Daphnia at 100% efficiency (Olmstead & Leblanc 2002). Due to the instability of MF, media was changed daily to ensure consistent exposure. The first brood was discarded and male neonates were collected from 2nd–3rd broods. Female and male cultures were maintained separately.
Maintained under standard condition with normal levels of oxygen (6 mg/L) for 3, 8 and 15 days.
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| Growth protocol |
Cultures of Daphnia pulex Eloise Butler strain (genotypes EB31 and EB45, originally sampled from Eloise Butler pond in Minnesota (Yampolsky et al. 2014)) 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. Oxygen concentrations were continuously monitored using an oxygen sensor (Unisense microrespiration system, Denmark). Daphnia samples were flash frozen in liquid nitrogen and stored in -80C until further use.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from the samples using MasterPure DNA purification kit (Epicentre, USA) following Athanasio et al. (2016).
Libraries for sodium bisulfite treated 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 pulex libraries were sequenced using Illumina NextSeq-500 platform at the Centre for Genomics and Bioinformatics, Indiana University, USA.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Bisulphite-treated genomic DNA
processed data file: annot_daphplx17evigenes_mod.gff.gz
processed data file: methylation_MethylKit_FvsM.txt.gz
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| Data processing |
Bisulfite-Seq: 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.
Bisulfite-Seq: The filtered reads were mapped to the reference genome of Daphnia pulex PA42 (GCA_900092285.1; Ye et al. 2017) using BWA Meth (v.0.10; Pedersen et al. 2014) for the bisulfite treated DNA samples, with default settings.
Bisulfite-Seq: The Daphnia pulex gene models used in the analysis are from November 2017 obtained from the arthropod database in eugenes (Genomic Information for Eukaryotic Organisms; http://arthropods.eugenes.org) produced by Don Gilbert using EvidentialGene (Gilbert 2016).
Bisulfite-Seq: Methylated CpG sites were called from mapped reads using MethylDackel (v.0.2.1; github.com/dpryan79/MethylDackel). Both uniquely mapped singletons and discordant reads were retained, while reads with low mapping quality (MAPQ < 10) and nucleotides with low base calling quality (Phred < 30) were excluded. Seven base pairs from both ends of the reads were also excluded, as they showed an excessive amount of methylation potentially due to adapter contamination.
Bisulfite-Seq: Differential methylation analysis was done using methylKit (v.1.3.0; Akalin et al. 2012). Akalin et al. 2012). CpG sites with abnormally high (>98 percentile) coverage were removed, as well as sites that were not covered in all samples or had zero methylated reads in more than half of the samples (n=6/12). Logistic regression was used to analyse differential CpG methylation between males and females, using genotype (EB31 and EB45) as a co-variable. The Q-values were adjusted using the SLIM method (Wang et al. 2011).
Genome_build: GCA_900092285.1
Supplementary_files_format_and_content: Bisulfite-Seq: bed (CpG-methylation calls from MethylDackel in methylKit format:*CpG.methylKit.gz)
Supplementary_files_format_and_content: Bisulfite-Seq: txt (differential CpG-methylation analysis results from methylKit: methylation_MethylKit_FvsM.txt.gz)
Supplementary_files_format_and_content: Bisulfite-Seq: gff (Daphnia pulex gene models used in the analysis are from November 2017 obtained from the arthropod database in eugenes (Genomic Information for Eukaryotic Organisms; http://arthropods.eugenes.org) produced by Don Gilbert using EvidentialGene (Gilbert 2016))
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| Submission date |
Dec 27, 2018 |
| Last update date |
Oct 18, 2019 |
| Contact name |
Jouni Antero Kvist |
| Organization name |
University of Helsinki
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| Department |
Research Program for Molecular Neurology
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| Lab |
Medical Neurogenetics group
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| Street address |
Haartmaninkatu 8
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| City |
Helsinki |
| State/province |
Uusimaa |
| ZIP/Postal code |
00014 |
| Country |
Finland |
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| Platform ID |
GPL24021 |
| Series (1) |
| GSE124427 |
A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex |
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| Relations |
| Reanalysis of |
GSM2786683 |
| BioSample |
SAMN10656453 |
| SRA |
SRX5185063 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3533026_GSF978-WGBS-F-45-2_S5_CpG.methylKit.txt.gz |
55.3 Mb |
(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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