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Reference: Hu, B, Petela, N, Kurze, A et al., (2015). Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq. Nucleic acids research, gkv670-gkv670.Citable link to this page:

 

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq.

Abstract: Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

Peer Review status:Peer reviewedPublication status:In pressVersion:Publisher's version Funder: Cancer Research UK   Notes:Copyright © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Bibliographic Details

Publisher: Oxford University Press

Publisher Website: http://www.oxfordjournals.org/

Journal: Nucleic acids researchsee more from them

Publication Website: http://nar.oxfordjournals.org/

Issue Date: 2015

pages:gkv670-gkv670Identifiers

Urn: uuid:988269ef-93ef-41eb-8050-37a248a5e2b6

Source identifier: 531420

Eissn: 1362-4962

Doi: https://doi.org/10.1093/nar/gkv670

Issn: 0305-1048 Item Description

Type: Journal article;

Language: eng

Version: Publisher's version Tiny URL: pubs:531420

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Autor: Hu, B - - - Petela, N - institutionUniversity of Oxford Oxford, MSD, Biochemistry - - - Kurze, A - institutionUniversity of Oxfor

Fuente: https://ora.ox.ac.uk/objects/uuid:988269ef-93ef-41eb-8050-37a248a5e2b6



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