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| Status |
Public on Apr 07, 2021 |
| Title |
O17 |
| Sample type |
SRA |
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| Source name |
telencephalon
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| Organism |
Taeniopygia guttata |
| Characteristics |
tissue: telencephalon
age: 65dph
Sex: M
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| Growth protocol |
For RRBS experiments, zebra finches (Taeniopygia guttata) were bred and raised at the University of Liège in an aviary containing 20 nest boxes maintained in a 13:11h light:dark cycle. Birds were provided ad libitum with food, water, grit and cuttlebone, and additional millet branches and egg food. Sprouted sunflower seeds were provided twice and nesting material once per week. The experiment was performed according to the Belgian law on animal experimentation and approved by the Ethical Committee for Animal Experimentation at the University of Liège (protocol 1396).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Brains from male and female juvenile and adult zebra finches (age: 1, 20, 65 (± 3 days) and adults (> 170) dph) were used, with birds from one nest randomly assigned to different age groups. Birds were rapidly decapitated, and the brain immediately removed and handled under ribonuclease-free conditions. The telencephalon was separated from the rest of the brain. The brain tissue was rapidly frozen on dry ice and stored at -80°C until further processing.
The combined interphase and organic (phenol) phase fractions of QiazolTM reagent-lysed samples were used for genomic DNA isolation. In short, ethanol was added to precipitate the DNA. The DNA pellet was then washed with sodium citrate solution (0.1M sodium citrate in 10% ethanol). Next, the pellet was incubated with 75% ethanol after which the pellet was dried and resuspended in DNase and RNase free water.
RRBS was performed by NXTGNT. Samples were purified using Zymo’s Genomic DNA Clean & ConcentratorTM-10 to avoid inhibition of enzymatic digestion. The Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen P11496) was used to assess the concentration of the samples. After pooling the samples per three in equal proportions, the resulting 24 pools were dried and redissolved in water (molecular grade) to obtain a concentration of 2 µg and subsequently digested using MspI (New England BioLabs R0106, 20 000 units/ml). After purification of the digestion product with a GeneJET PCR Purification Kit (ThermoFisher K0701), the Qubit 2.0 Fluorometer (Q32866) was used to determine the concentration.
For the library preparation, the NEBNext® UltraTM DNA Library Prep Kit (New England BioLabs, E7370S) was used. For each sample, half of the DNA was oxidized for oxRRBS, while the other half was subjected to regular RRBS analysis. Subsequently, bisulfite and oxidative bisulfite conversion were performed using the TrueMethyl Seq kit (Cambridge Epigenetix, CEGX). A PCR with TrueMethyl DNA Polymerase was performed as a bisulfite conversion control and showed no aberrations. RRBS sequencing was performed on the Illumina NextSeq500. As initial paired-end experiments (2 x 76nt) demonstrated low quality of reverse reads, only forward reads were used, and subsequent experiments relied on single-end sequencing (1 x 76nt). Also, low-diversity issues led to overclustering and lower sequencing efficiency. Therefore, in collaboration with Illumina, different numbers of dark cycles were used to optimize the resulting useable library size (see Supplementary Information Table S1). Additionally, different concentrations of control PhiX were used as a function of other spiked-in libraries (independent of this project). Given that samples were randomized over different sequencing pools and the optimization particularly focused on increasing yield (forward read data quality was similar and acceptable for all experiments), no bias and at most a limited increase in technical variance is expected due to these variations in the protocol.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For RRBS, quality control and filtering of low quality reads was performed using “Trim Galore!” (Babraham Bioinformatics). Quality control indicated no relevant problems, so Bismark (v0.9.0, Babraham Bioinformatics) was used in Bowtie2-mode [21] for mapping. Seed length, mismatches and interval during multiseed alignment were set to the default values. The data were subsequently imported using the Bioconductor BiSeq package [22] for extraction and summarization of methylation values per CpG, CHH and CHG. The total methylation counts were used as proxy for DNA methylation since hydroxymethylation was negligible and methylation-specific results were featured by a too low coverage for several samples (see results). Ensembl (assembly taeGut3.2.4, release 87) and Uniprot annotation (R-package UniProt.ws, v2.14.0) were used for annotation of transcription start site (TSS), start and end positions of genes and obtaining gene symbols for ensembl gene ids throughout all analyses.
Genome_build: taeGut3.2.4
Supplementary_files_format_and_content: .bismark.cov
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| Submission date |
Apr 02, 2020 |
| Last update date |
Apr 07, 2021 |
| Contact name |
Jeroen Galle |
| Organization name |
Ghent University
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| Department |
Dept. Of Mathematical Modelling, Statistics and Bioinformatics
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| Lab |
Biobix, Lab of Bioinformatics and Computational Genomics
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| Street address |
Coupure Links 653
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| City |
Ghent |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL22780 |
| Series (2) |
| GSE147973 |
DNA methylation regulates transcription factor specific neurodevelopmental but not sexually dimorphic gene expression dynamics in zebra finch telencephalon [RRBS] |
| GSE147974 |
DNA methylation regulates transcription factor specific neurodevelopmental but not sexually dimorphic gene expression dynamics in zebra finch telencephalon |
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| Relations |
| BioSample |
SAMN14525650 |
| SRA |
SRX8047075 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4451207_CpG_context_oxRRBS_ZF_brain.O17_R1_cleanSE.fastq.gz_bismark_bt2.bismark.cov.gz |
7.3 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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