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A method for assessing histone surface accessibility genome-wide.

Methods (San Diego, Calif.) (2019-12-13)
Luke T Marr, David J Clark, Jeffrey J Hayes, Luke T Marr, David J Clark, Jeffrey J Hayes
RÉSUMÉ

The assembly of DNA into nucleosomes and higher order chromatin structures serves not only as a means of compaction but also organizes the genome to facilitate crucial processes such as cell division and regulation of gene expression. Chromatin structure generally limits access to DNA, with the accessibility of DNA in chromatin being regulated through post translational modification of the histone proteins as well as the activity of chromatin remodeling proteins and architectural chromatin factors. There is great interest in assessing chromatin accessibility genome-wide to identify functional elements associated with enhancers, promoters, and other discontinuities in the compacted chromatin structure associated with gene expression. As the vast majority of techniques rely upon assessment of the exposure of the underlying DNA, we describe here a general method that can be used to assess exposure of internal and external histone protein surfaces. We demonstrate the feasibility of this method, in the organism S. cerevisiae. Our method relies on substitution of residues residing on selected histone protein surfaces with cysteine, and assessment of exposure by reaction with a thiol specific reagent, biotin-maleimide. We demonstrate that modified nucleosomes can be efficiently excised from nuclei treated with the reagent via a one-step purification process. After library preparation and deep sequencing, selected nucleosomes are typically ~25-fold enriched over background signals and exhibit phasing with respect to transcription start sites in yeast that is identical to an unselected population.

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Description du produit

Sigma-Aldrich
2-mercaptoéthanol, ≥99.0%
Sigma-Aldrich
Lyticase from Arthrobacter luteus, lyophilized powder, ≥2,000 units/mg protein, Protein ≥20 % by biuret
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Maleimide, 99%
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Glass beads, diam. 1.0 mm, for Beadbeating
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DL-Cysteine, technical grade