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| Status |
Public on Sep 06, 2016 |
| Title |
RatID_5_MBD |
| Sample type |
SRA |
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| Source name |
dorsal hippocampus, sham operated
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
gender: male
tissue: dorsal hippocampus
treatment: sham operated
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| Treatment protocol |
Starting from the day of surgery each animal was housed in a separate cage. Surgery was performed under isoflurane anesthesia (2-2.5% in 100% O2) preceded by the injection of butorphanol (Butomidor, Richter Pharma AG, Wells, Austria; 0.5 mg/kg, i.p.). A stimulating and recording bipolar wire electrode (Plastic One Inc., Roanoke, VA, #E363-3-2WT-SPC) were implanted into the left lateral nucleus of the amygdala (AP -3.6 mm, L 5.0 mm from the bregma; DV 6.5 mm from the surface of the brain. A stainless stell screw electrode (Plastic One Inc., Roanoke, VA, #363/20) was implanted contralaterally into the skull over the right frontal cortex (AP +3.0 mm, L 2.0 mm from the bregma) as a surface EEG recording electrode. Two stainless steel screw electrodes were placed bilaterally over the cerebellum (AP -10.0 mm, L 2.0 mm from the bregma) as ground and reference electrodes. The socket contracts of all electrodes were placed in a multi-channel electrode pedestal (Plastic One Inc., Roanoke, VA, #MS363) which was attached to the skull with dental acrylate (Duracryl Plus). After 2 weeks of recovery, animals were electrically stimulated via the intra-amygdala electrode to evoke status epilepticus (SE). Stimulation consisted of a 100-ms train of 1-ms biphasic square-wave pulses (400µA peak to peak) delivered at 60Hz every 0.5 s for 30 min. If the animal did not enter SE, stimulation was continued for an additional 10 min. The SE was stopped 1.5-2h after stimulation via an intraperitoneal injection of diazepam (20 mg/kg; Relanium, Polfa, Warsaw). If the first dose of diazepam did not suppress SE, the animal received subsequent doses of diazepam (5mg/kg). Sham operated controls were aged matched and had electrodes but did not receive electrical stimulation. Stimulated rats were monitored with video EEG (Comet EEG, Grass Technologies, West Warwick, RI) for 2 weeks before the end of experiment to determine the presence of spontaneous seizures. Spontaneous seizures were identified from EEG recording by browsing the EEG manually. An electrographic seizure was defined as high frequency (> 8Hz), high amplitude (>2x baseline) discharge lasting for at least 5s. All stimulated animals enrolled in the study had spontaneous seizures.
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| Growth protocol |
Male Sprague-Dawley (Medical Research Centre, Warsaw, Poland) weight 290-320 g were housed in a controlled enviroment (21-23 °C, 12h dark/light cycles) with drinking and feeding ad libitum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was collected at 3 months after induction of status epilepticus. For tissue collection rats were anesthetized with CO2 and decapitated with guillotine. Left hippocampus was rapidly isolated and tissue containing CA3 and dentate gyrus was homogenized in ice cold 1x PBS and divided into equal volumes for DNA and RNA extraction. Samples were stored at -80oC until use.
2000 ng of hippocampal DNA from each animal was fragmented to median size of 200-300 bp and subjected to methylated DNA capture according to MethylMiner protocol (Invitrogen, Darmstadt, Germany), exclusively enabling capture of methylated double stranded DNA. Fragmented and enriched DNA was eluted at high salt concentrations (2M NaCl). 5ng of enriched DNA was used in library preparation using the NEBNext DNA Library Prep Reagent Set for Illumina (New England Biolabs, Frankfurt/Main, Germany). Quality of sequencing libraries was assayed using the Shimadzu MultiNA capilary electrophoresis system (Shimadzu, Kyoto, Japan). Libraries were sequenced at a concentration of 10 pM on the Illumina Genome Analyzer IIx (Illumina, San Diego, CA, USA) with a 36 bp single read length.
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| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
MBD_RID_5_C_ASE
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| Data processing |
Image analysis and base calling were performed with OLBv1.8 software.
Reads were aligned to the rat genome (build rn4) using BWA (aln algorithm v0.5.9) with default settings.
Profiles of DNA methylation were compared between all pairwise combinations of samples for each model separately using the MACS peak calling software (version 1.4.0 rc2) with fixed shift size of 75 bp and a significance cut-off 10E-05.
Genomic regions showing different methylation patterns between pairs of samples were merged using BEDTools.
Duplicate reads which aligned to the same location in a given sample were removed from futher analysis. The numbers of read tags aligning to each region were summarized using custom python script producing a matrix of counts (tags per region per sample). The regions were non-differentially filtered for regions, where the sum of tag counts was below the 50th centile.
Genome_build: RGSC_v3.4
Supplementary_files_format_and_content: Tab-delimited count matrix.
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| Submission date |
Nov 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Katarzyna Lukasiuk |
| E-mail(s) |
k.lukasiuk@nencki.gov.pl
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| Organization name |
Nencki Institute of Experimental Biology Polish Academy of Sciences
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| Department |
Department of Molecular and Cellular Neurobiology
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| Lab |
Laboratory of Epileptogenesis
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| Street address |
3 Pasteur Street
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| City |
Warsaw |
| ZIP/Postal code |
02-093 |
| Country |
Poland |
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| Platform ID |
GPL10669 |
| Series (2) |
| GSE75400 |
Etiology matters - Comparing Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy (Amygdala stimulation – MBD-seq) |
| GSE75403 |
Etiology matters - Comparing Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy |
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| Relations |
| BioSample |
SAMN04295914 |
| SRA |
SRX1452248 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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