Locus-specific DNA methylation analysis of retrotransposons in ES, somatic and cancer cells using High-Throughput Targeted Repeat Element Bisulfite Sequencing.

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In order to study DNA methylation of a large number of retrotransposons on an individual-locus basis, we have developed a new protocol termed High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS) (Ekram and Kim, 2014 [1]). We have used this technique to characterize the locus-specific patterns of DNA methylation of 4799 members of the mouse IAP LTR (Intracisternal A Particle Long Terminal Repeat) retrotransposon family in embryonic stem, somatic and Neuro2A cells (Bakshi and Kim, 2014 [2]). Here we describe in detail the sample preparation and bioinformatics analyses used for these studies. The somatic cell data may be accessed under GEO accession number GSE49222. The ES and Neuro2A data are deposited under GEO accession number GSE60007.", "claims": [{"id": "claim-data-access", "statement": "Raw and processed data for the studies described here may be found at the following links:\nSomatic tissue: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49222\nES and Neuro2A cells: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60007", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:25554740", "locator": "sections[2] \"Direct link to deposited data\" paragraphs 1-4", "quote": "Raw and processed data for the studies described here may be found at the following links:\nSomatic tissue: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49222\nES and Neuro2A cells: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60007"}], "state": "complete"}, {"id": "claim-target-result", "statement": "The study characterized locus-specific DNA methylation patterns for 4799 mouse IAP LTR retrotransposons using HT-TREBS, with BiQ Analyzer HT for methylation analysis and deposited raw and processed data available through GEO accessions GSE49222 and GSE60007.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25554740", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We have used this technique to characterize the locus-specific patterns of DNA methylation of 4799 members of the mouse IAP LTR (Intracisternal A Particle Long Terminal Repeat) retrotransposon family in embryonic stem, somatic and Neuro2A cells (Bakshi and Kim, 2014 [2])."}, {"asset": "source:pmid:25554740", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The somatic cell data may be accessed under GEO accession number GSE49222. The ES and Neuro2A data are deposited under GEO accession number GSE60007."}, {"asset": "source:pmid:25554740", "locator": "sections[5] \"Bioinformatics analyses\" paragraph 2", "quote": "We have used Bowtie2 [3] for mapping and BiQ analyzer HT [4] for DNA methylation analyses."}, {"asset": "source:pmid:25554740", "locator": "sections[2] \"Direct link to deposited data\" paragraph 2", "quote": "Raw and processed data for the studies described here may be found at the following links:"}, {"asset": "source:pmid:25554740", "locator": "sections[2] \"Direct link to deposited data\" paragraph 3", "quote": "Somatic tissue: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49222"}, {"asset": "source:pmid:25554740", "locator": "sections[2] \"Direct link to deposited data\" paragraph 4", "quote": "ES and Neuro2A cells: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60007"}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The study characterized locus-specific DNA methylation patterns for 4799 mouse IAP LTR retrotransposons using HT-TREBS, with BiQ Analyzer HT for methylation analysis and deposited raw and processed data available through GEO accessions GSE49222 and GSE60007.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:25554740"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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We hav", "olated DNA was sonicated using the Bioruptor NGS (Diagenode) to obtain fragments which were a", "was used for the emulsion PCR and subsequent next-generation sequencing using the Ion Torrent Personal Genome Machin"], "order_basis": "paper_text_order:69", "id": "assay:1"}, {"role": "data_generation", "definition_id": "methylation_array", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["sing High-Throughput Targeted Repeat Element Bisulfite Sequencing.", "rmed High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS) (Ekram and Kim, 2014 [", ") and subsequently subjected to one round of bisulfite conversion using the EZ DNA Methylation Kit", "f size selection, all fragments > 300 bp are bisulfite treated and PCR amplified. The cycle number"], "order_basis": "paper_text_order:93", "id": "assay:2"}, {"role": "data_generation", "definition_id": "animal_model", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["ns of DNA methylation of 4799 members of the mouse IAP LTR (Intracisternal A Particle Long Term", "r and kidney from a 1-week-old male C57BL6/N mouse were lysed overnight in 10X w/v Tail Lysis B"], "order_basis": "paper_text_order:595", "id": "assay:3"}, {"role": "data_generation", "definition_id": "qpcr", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["tial portion of the amplification curve from qPCR. Finally, the amplified library was size sel", "(1 μL) was then used to conduct quantitative real-time PCR using SYBR-Green (Life Technologies) to dete"], "order_basis": "paper_text_order:3612", "id": "assay:4"}]}