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Sample GSM2390019 Query DataSets for GSM2390019
Status Public on May 01, 2017
Title input_64cell_rep1
Sample type SRA
 
Source name Embryos
Organism Danio rerio
Characteristics developmental stage: 64-cell embryos
strain: AB wild type
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.
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).
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description RIP antibody: none
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
 
Submission date Nov 14, 2016
Last update date May 15, 2019
Contact name Håvard Aanes
E-mail(s) Havard.Aanes@rr-research.no
Organization name Oslo University Hospital
Department Dept of microbiology
Lab Klungland Lab
Street address Sognsvannsveien 20
City Oslo
ZIP/Postal code 0372
Country Norway
 
Platform ID GPL14875
Series (1)
GSE89815 N6-methyladenosine dynamics during early vertebrate embryogenesis
Relations
BioSample SAMN06014717
SRA SRX2345561

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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