Fragmentation ensures that protein/DNA complexes in high–molecular-weight chromatin are soluble and accessible to your ChIP antibody. The method of fragmentation that you choose should depend on whether you performed N-ChIP or X-ChIP. If you are performing N-ChIP, then you should fragment the DNA with appropriate enzymes (such as micrococcal nuclease), because mechanical shearing methods will disrupt native histone/DNA associations. If you are performing X-ChIP, you may choose either mechanical sonication or enzymatic digestion to fragment your DNA. In either case it is important to optimize shearing conditions and use those exact same conditions for each experiment to reduce the potential for variability in the starting chromatin (Figure 1).
Figure 1.Agarose gel electrophoresis analysis of purified DNA fragments. The purified DNA fragments have undergone sonication (Lane 2) or no sonication (Lane 1), proteinase digestion, crosslink reversal, extraction and precipitation. We recommend that you analyze the DNA on an agarose gel after each sonication experiment.
NOTE: that heterochromatin may be resistant to sonication and this may reduce the yield of chromatin.
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