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Genome-wide profiling of DNA methyltransferases in mammalian cells


Manzo, Massimiliano; Ambrosi, Christina; Baubec, Tuncay (2018). Genome-wide profiling of DNA methyltransferases in mammalian cells. In: Vavouri, Tanya; Peinado, Miguel A. CpG Islands. Berlin, Germany: Springer, 157-174.

Abstract

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.

Abstract

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.

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Item Type:Book Section, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:570 Life sciences; biology
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Uncontrolled Keywords:ChIP-seq, CpG islands, DNA methyltransferases, Immunoprecipitation, In vivo biotinylation, Next-generation sequencing
Language:English
Date:2018
Deposited On:09 Aug 2018 15:08
Last Modified:29 Jul 2020 07:10
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:1766
ISSN:1064-3745
ISBN:978-1-4939-7767-3
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-4939-7768-0_9
Related URLs:https://www.recherche-portal.ch/primo-explore/fulldisplay?docid=ebi01_prod011179482&context=L&vid=ZAD&search_scope=default_scope&isFrbr=true&tab=default_tab&lang=de_DE (Library Catalogue)
PubMed ID:29605852

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