Published in
BioTechniques Protocol Guide 2007
(p.57)
Abstract
The Affymetrix Chromatin Immunoprecipitation Protocol is a complete start-to-finish protocol to prepare samples for chromatin immunoprecipitation experiments with microarrays.
The Affymetrix Chromatin Immunoprecipitation Protocol is a complete start-to-finish protocol to prepare samples for chromatin immunoprecipitation experiments with microarrays. Using the powerful combination of chromatin immunoprecipitation and GeneChip® Tiling Arrays, it is possible to map the interaction of proteins and DNA on a genome-wide scale.
This comprehensive protocol includes a unique amplification and digestion method to ensure that DNA fragments are uniformly sized for reproducible array hybridizations. The protocol is compatible with GeneChip® Tiling Arrays for human, mouse, Arabidopsis, C. elegans, S. pombe, and S. cerevisiae.
As highlighted in Figure 1, cells are first fixed with formaldehyde to crosslink DNA to any associated proteins (A). The cells are then lysed and DNA is sheared into smaller fragments using sonication (B). Protein-DNA complexes are then immunoprecipitated with an antibody directed against the specific protein of interest (C). Following the immunoprecipitation crosslinking is reversed and samples are protease-treated (D). The purified DNA sample is then amplified using a random-primed PCR method (E). Subsequently, targets are fragmented and labeled (F) and hybridized (G) to GeneChip® Tiling Arrays. Hybridization signals generated by control and experimental samples are compared and the regions of protein-DNA interaction can be identified.
Figure 1. (Click to enlarge)
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