DNA unmethylome profiling by covalent capture of CpG sites
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE48994 | GEO | 2013/08/27
SECONDARY ACCESSION(S): PRJNA212537
REPOSITORIES: GEO
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Project description:Here we present an approach for the mapping of unmethylated DNA. Our technique is based on DNA methyltransferase-directed transfer of activated groups (mTAG) and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interogation on tiling microarrays or next generation sequencing.
2013-08-27 | GSE49044 | GEO
Project description:Here we present an approach for the mapping of unmethylated DNA. Our technique is based on DNA methyltransferase-directed transfer of activated groups (mTAG) and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interogation on tiling microarrays or next generation sequencing. mapping of unmethylated DNA
2013-08-27 | E-GEOD-49044 | biostudies-arrayexpress
Project description:Here we present an approach for the mapping of unmethylated DNA. Our technique is based on DNA methyltransferase-directed transfer of activated groups (mTAG) and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interogation on tiling microarrays or next generation sequencing. mTAG labeled genomic DNA of one sample
2013-08-27 | E-GEOD-48994 | biostudies-arrayexpress
Project description:Cytosine 5-modification is a widespread epigenetic mark in eukaryote genomes including humans, but its large scale populational studies are hampered by substantial limitations of the existing analytical techniques. We have developed a new approach for mapping of the unmodified fraction of the genome, i.e. DNA unmethylome, which is based on covalent tagging of unmodified CpG sites with biotin. Sequence-specific tagging of DNA was achieved by using an engineered version of the SssI cytosine-5 methyltransferase and a synthetic analog of the S-adenosylmethionine cofactor carrying a transferable 6-aminohex-2-ynyl group. Analysis of the streptavidin-enriched human unmethylome on tiling DNA microarrays showed that the new approach (named mTAG-chip) offers nanogram sensitivity, a substantially higher precision in the low and medium CpG density regions and added versatility as compared with the existing epigenome profiling techniques.
2012-03-08 | GSE36305 | GEO
Project description:Cytosine 5-modification is a widespread epigenetic mark in eukaryote genomes including humans, but its large scale populational studies are hampered by substantial limitations of the existing analytical techniques. We have developed a new approach for mapping of the unmodified fraction of the genome, i.e. DNA unmethylome, which is based on covalent tagging of unmodified CpG sites with biotin. Sequence-specific tagging of DNA was achieved by using an engineered version of the SssI cytosine-5 methyltransferase and a synthetic analog of the S-adenosylmethionine cofactor carrying a transferable 6-aminohex-2-ynyl group. Analysis of the streptavidin-enriched human unmethylome on tiling DNA microarrays showed that the new approach (named mTAG-chip) offers nanogram sensitivity, a substantially higher precision in the low and medium CpG density regions and added versatility as compared with the existing epigenome profiling techniques. AC103, AC13 and AC31 are DNA samples from three postmortem brains. Nr1 and Nr2 are two replicates of each sample. 5%, 20% and 70% are labeling efficiency. K means 0% labeling efficiency. We were testing which labeling efficiency was best by comparing to published seq data using correlation.
2012-03-08 | E-GEOD-36305 | biostudies-arrayexpress
Project description:We describe the development and thorough validation of a new approach for epigenome analysis (named TOP-seq) that is based on a novel concept – combination of selective covalent tagging and targeted genomic sequencing of the genome. The technique reports the modification status of each genomic CpG site by priming the DNA polymerase action from a tethered oligonucleotide probe that is selectively attached to the unmodified CpG sites.
2017-01-16 | GSE91023 | GEO
Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray
2010-05-27 | GSE19788 | GEO
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.
2015-09-03 | GSE60988 | GEO
Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.
2010-05-27 | E-GEOD-19788 | biostudies-arrayexpress
Project description:Following erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at the time of implantation in each individual through a wave of generalized de novo modification with CpG islands specifically left unmethylated. In this paper, we ask whether CpG islands are M-bM-^@M-^\protectedM-bM-^@M-^] from de novo methylation or, alternatively, acquire their unmodified state through M-bM-^@M-^\demethylation.M-bM-^@M-^] Using a genetic approach, we demonstrate that ES cells indeed have the ability to specifically demethylate CpG islands through pathways involving hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not actually necessary for generating the overall bimodal methylation pattern in ES cells. This general activity does, however, appear to be involved both in somatic cell reprogramming, as well as the normal methylation reprogramming that takes place in the early embryo. CpG-methylated genomic DNA was enriched using a methyl-DNA immunoprecipitation (mDIP) assay. DNA from the input and bound (enriched) DNA for each sample were labeled and hybridized on the array to define the methylation state of each region.
2013-11-17 | E-GEOD-48923 | biostudies-arrayexpress