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Status |
Public on May 01, 2017 |
Title |
input_64cell_rep1 |
Sample type |
SRA |
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Source name |
Embryos
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Organism |
Danio rerio |
Characteristics |
developmental stage: 64-cell embryos strain: AB wild type
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Growth protocol |
Embryos from the AB wild type strain were obtained from the NMBU zebrafish facility where the zebrafish were kept at 28±1°C on a 14-10 hour light-dark cycle at a density of 5-10 fish/L. System water (SW) was prepared from particle and active charcoal filtrated tap water, deionized by reverse osmosis (RO) and kept sterile by UV irradiation. The water was conditioned to a conductivity of 500µS/cm, general hardness (GH) 4-5 and pH 7.5 by addition of 155g synthetic sea salt (Instant Ocean, Blacksburg, USA), 53g sodium carbonate and 15g calcium chloride (Sigma-Aldrich) per liter RO water. Adult fish were fed with Gemma Micro 300 (Skretting, Stavanger, Norway) dry feed twice a day and live artemia (Scanbur, Karlslunde, Denmark) once a day. Health monitoring was by daily inspection, use of sentinel fish sent for pathology (ZIRC, Eugene, Oregon) and water microbiology analysis (NMBU Vetbio, Oslo) every six month. Adult fish were allowed to mate for 30 minutes in standard 1L breeding tanks (Aquatic Habitats, Apopka, FL). Harvested embryos were kept in autoclaved SW at 28 °C, harvested by snap freezing (liquid nitrogen) and visually controlled for stage and lack of abnormalities at the selected time points. All experiments were performed according to Norwegian Animal Welfare Act (2009), the EU Directive 2010/63.
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Extracted molecule |
total RNA |
Extraction protocol |
We isolated total RNA from 4 stages (1-cell/20 minutes post fertilization, 2-, 4- and 6-hpf) in batches of 200 embryos using TRIzol (Invitrogen, cat.no. 15596-018). We added ERCC spike-in RNA (TermoFisher scientific, cat.no. 4456740) to the trizol. For each sequencing library two batches of total RNA (from 200 embryos) were merged and enriched for polyA+ RNA using Dynabeads® mRNA Purification Kit (Ambion, #61006). The m6A-RIP experiment was carried out as previously described (Ke et al., 2015) and the protocol can be found in supplementary file 1. Briefly, polyA+ enriched RNA was partially fragmented by alkaline hydrolysis, ethanol precipitated and SDS-PAGE size selected for 20-80nt. Part of the fragmented RNA was used as input and the rest was immunoprecipitated at 4oC for 2 h using Dynabeads Protein A (Life Technologies, # 10008D)-conjugated anti-m6A antibody (Synaptic systems, # 202003). After stringent washing, the bound RNA was eluted with 0.5mg/ml N6-methyladenosine sodium salt (Sigma-Aldrich, # M2780), ethanol precipitated, and resuspended with RNase-free water. The eluted RNA and input RNA were subjected to 3’pre-adenylated DNA liker ligation with T4 RNA ligase2, truncated KQ (NEB, #M0373L), at 16 oC over night. The sequencing library was constructed with bromodeoxyuridine (BrdU)-CLIP protocol described in Weyn-Vanhentenryck et al. (2014), with improved RT primers indicated in Ke et al. (2015).
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
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Description |
RIP antibody: none
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Data processing |
Base calling The sequencing output from each of the 4 lanes the sequencing run was trimmed 7 nucleotides in the 5’end, and de-multiplexed (4 samples in each lane). Reads were mapped to Zv10 using the STAR aligner (Dobin et al., 2013) with options --seedSearchStartLmax 15 --clip3pNbases 10 --clip5pNbases 10 --outFilterMultimapNmax 20 --outFilterMismatchNoverLmax 0.05 --outFilterMatchNminOverLread 0.0 --outFilterMatchNmin 15 --outFilterScoreMinOverLread 0.0. We performed peak calling and detection of differentially methylated genes using ExomePeak (Meng et al., 2013). Genome_build: Zv10 Supplementary_files_format_and_content: Bed files with enriched regions from each developmental stage, and txt file with raw and normalized read counts for each gene using the input samples
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Submission date |
Nov 14, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Håvard Aanes |
E-mail(s) |
Havard.Aanes@rr-research.no
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Organization name |
Oslo University Hospital
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Department |
Dept of microbiology
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Lab |
Klungland Lab
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Street address |
Sognsvannsveien 20
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City |
Oslo |
ZIP/Postal code |
0372 |
Country |
Norway |
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Platform ID |
GPL14875 |
Series (1) |
GSE89815 |
N6-methyladenosine dynamics during early vertebrate embryogenesis |
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Relations |
BioSample |
SAMN06014717 |
SRA |
SRX2345561 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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