Nucleic Acids Research Advance Access originally published online on September 2, 2008
Nucleic Acids Research 2008 36(19):e125; doi:10.1093/nar/gkn535
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Nucleic Acids Research, 2008, Vol. 36, No. 19 e125
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
*To whom correspondence should be addressed. Tel: +1 617 432 2104; Fax: +1 617 432 2529; Email: kevin{at}hms.harvard.edu
Received June 13, 2008. Revised July 31, 2008. Accepted August 4, 2008.
Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.