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Sci Rep. 2014 Jun 12;4:5152. doi: 10.1038/srep05152.

Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: identifying ChIP-quality p300 monoclonal antibodies.

Author information

1
1] Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA [2].
2
Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA.
3
HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.

Abstract

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1.

PMID:
24919486
PMCID:
PMC4053718
DOI:
10.1038/srep05152
[Indexed for MEDLINE]
Free PMC Article

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