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. 2011 Sep 29;6(10):1656-68.
doi: 10.1038/nprot.2011.402.

Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq

Mazhar Adli  1 , Bradley E Bernstein
Affiliations

Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq

Mazhar Adli et al. Nat Protoc. .

Abstract

Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP protocols necessitate the use of large numbers of cells, and library preparation steps associated with current high-throughput sequencing platforms require substantial amounts of DNA; both of these factors preclude the application of ChIP-seq technology to many biologically important but rare cell types. Here we describe a nano-ChIP-seq protocol that combines a high-sensitivity small-scale ChIP assay and a tailored procedure for generating high-throughput sequencing libraries from scarce amounts of ChIP DNA. In terms of the numbers of cells required, the method provides two to three orders of magnitude of improvement over the conventional ChIP-seq method and the entire procedure can be completed within 4 d.

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Figures

Figure 1
Figure 1
Schematic of nano-ChIP-seq library preparation method. Fragmented ChIP DNA is primed (stage A) with Sequenase enzyme using custom designed primer 1 that contains universal sequence (green), the BciVI restriction site (red) and random 9-mer (blue). Primed ChIP DNA is then amplified using primer 2 that contains the same universal sequence and the restriction site as in primer 1 (stage B). Finally, BciVI digestion of amplified templates generates DNA fragments bearing 3′-A overhangs on both ends that can be ligated with standard Illumina adapters (stage C). RE, restriction enzyme.
Figure 2
Figure 2
The sonication setup with the Branson 250. The Eppendorf tube is secured with the help of a clamp so that the probe does not touch the side or the bottom of the tube. During sonication, the tube is kept on wet ice to minimize denaturation and dissociation of proteins from chromatin.
See this image and copyright information in PMC

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