Document Detail


ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy.
MedLine Citation:
PMID:  23026909     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
This unit describes the ChIP-exo methodology, which combines chromatin immunoprecipitation (ChIP) with lambda exonuclease digestion followed by high-throughput sequencing. ChIP-exo allows identification of a nearly complete set of the binding locations of DNA-binding proteins at near-single-nucleotide resolution with almost no background. The process is initiated by cross-linking DNA and associated proteins. Chromatin is then isolated from nuclei and subjected to sonication. Subsequently, an antibody against the desired protein is used to immunoprecipitate specific DNA-protein complexes. ChIP DNA is purified, sequencing adaptors are ligated, and the adaptor-ligated DNA is then digested by lambda exonuclease, generating 25- to 50-nucleotide fragments for high-throughput sequencing. The sequences of the fragments are mapped back to the reference genome to determine the binding locations. The 5' ends of DNA fragments on the forward and reverse strands indicate the left and right boundaries of the DNA-protein binding regions, respectively.
Authors:
Ho Sung Rhee; B Franklin Pugh
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]     Volume:  Chapter 21     ISSN:  1934-3647     ISO Abbreviation:  Curr Protoc Mol Biol     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-02     Completed Date:  2013-01-30     Revised Date:  2013-11-07    
Medline Journal Info:
Nlm Unique ID:  8908160     Medline TA:  Curr Protoc Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  Unit 21.24     Citation Subset:  IM    
Copyright Information:
2012 by John Wiley & Sons, Inc.
Affiliation:
Pennsylvania State University, University Park, Pennsylvania, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Binding Sites
Chromatin Immunoprecipitation / methods*
DNA-Binding Proteins / metabolism*
Genome*
High-Throughput Nucleotide Sequencing / methods*
Humans
Protein Binding
Reproducibility of Results
Saccharomyces cerevisiae / genetics,  metabolism
Sequence Analysis, DNA / methods*
Grant Support
ID/Acronym/Agency:
ES013768/ES/NIEHS NIH HHS; R01 ES013768/ES/NIEHS NIH HHS; R01 GM059055/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/DNA-Binding Proteins
Comments/Corrections

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