Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published June 12, 2014 | Published + Supplemental Material
Journal Article Open

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


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.

Additional Information

© 2014 Macmillan Publishers Limited. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. The images in this article are included in the article's Creative Commons license, unless indicated otherwise in the image credit; if the image is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the image. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Received 26 March 2014; Accepted 13 May 2014; Published 12 June 2014. The authors thank Henry Amrhein, Diane Trout and Sean Upchurch for technical and computational assistance, Dan Savic for discussions and contributions to our manual ChIP production and development efforts and the Caltech Protein Expression Center and Monoclonal Antibody Facility for production of monoclonal antibodies. This work was supported by the Beckman Institute Functional Genomics Center, the Donald Bren Endowment, and NIH grants U54 HG004576 and U54 HG006998. Author contributions: B.J.W., J.V. and W.C.G. designed the R-ChIP protocol and experiments to test it; W.C.G., J.V. and M.T.S. established and refined the protocol and performed the R-ChIP experiments; F.P. and K.N. developed and carried out all ChIP library construction for sequencing and executed comparative manual ChIP, G.D. and J.V. designed and screened, and S.O. immunized and produced the p300 monoclonal antibodies; G.K.M. performed computational analyses; G.K.M., W.C.G., J.V., F.P., R.M.M. and B.J.W. wrote the paper.

Attached Files

Published - srep05152.pdf

Supplemental Material - srep05152-s1.pdf


Files (5.4 MB)
Name Size Download all
2.3 MB Preview Download
3.1 MB Preview Download

Additional details

August 22, 2023
October 26, 2023