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A streamlined method for rapid and sensitive chromatin immunoprecipitation.
Sikes, Michael L; Bradshaw, Justin M; Ivory, Wendell T; Lunsford, Jessica L; McMillan, Ruth E; Morrison, Clayton R.
J Immunol Methods ; 344(1): 58-63, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19328803

ABSTRACT

We report a streamlined procedure to efficiently carry samples from chromatin to qPCR-compatible DNA in as little as 4 h. We use this streamlined ChIP to quantify histone H3 modifications at active (cad) and repressed (T early alpha) promoters in a Rag1-deficient pro-T cell line after 1-2 h IP. We further show that the protocol readily quantified histone modifications in chromatin from 10(4) Rag-deficient DN thymocytes. Taken together, these data outline a simple, cost-effective procedure for efficient ChIP analysis.


Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/isolation & purification , Histones/metabolism , Animals , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Cell Line , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dihydroorotase/genetics , Dihydroorotase/metabolism , Gene Knockdown Techniques , Histones/genetics , Mice , Mice, Knockout , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , T-Lymphocytes/metabolism
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