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Nucleic Acids Res. 2013 Apr;41(7):e87. doi: 10.1093/nar/gkt081. Epub 2013 Feb 14.

High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

Author information

1
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.

Abstract

We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. We achieved ≈ 10 000-fold enrichment of specific loci, which enabled sequencing nucleosomes at up to ≈ 500-fold higher coverage than has been reported in a mammalian genome. We demonstrate the advantages of generating high-sequencing coverage for mapping the center of discrete nucleosomes, and we show the use of the method by mapping nucleosomes during T cell differentiation using nuclei from effector T-cells differentiated from clonal, isogenic, naïve, primary murine CD4 and CD8 T lymphocytes. The analysis reveals that discrete nucleosomes exhibit cell type-specific occupancy and positioning depending on differentiation status and transcription. This method is widely applicable to mapping many features of chromatin and discerning its landscape in large genomes at unprecedented resolution.

PMID:
23413004
PMCID:
PMC3627574
DOI:
10.1093/nar/gkt081
[Indexed for MEDLINE]
Free PMC Article

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