Abstract
Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.
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Acknowledgements
We thank H. Keren-Shaul for important comments and G. Brodsky for artwork. This project was supported by an excellence grant from the Weizmann institute (R.B.-G.), The Human Frontiers Science Program, Career Development Award; an Israel Science Foundation (ISF) Bikura Institutional Research Grant Program; ERC starting grant 309788; and the Center for Excellence in Genome Science from the National Human Genome Research Institute (NHGRI) 1P50HG006193 (I.A.).
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R.B.-G. and I.A. designed the experiments. R.B.-G., D.J., D.A.-Z., D.L.-A. and I.A. conducted the experimental work. Z.B.-I. performed the computational analysis. R.B.-G., D.J., Z.B.-I. and I.A. wrote the manuscript.
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Supplementary information
Supplementary Table 1
ChIP-grade antibodies used in this manuscript (PDF 269 kb)
Supplementary Table 2
Sequencing library barcodes (PDF 215 kb)
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Blecher-Gonen, R., Barnett-Itzhaki, Z., Jaitin, D. et al. High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat Protoc 8, 539–554 (2013). https://doi.org/10.1038/nprot.2013.023
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DOI: https://doi.org/10.1038/nprot.2013.023
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