Chromatin Fragmentation

After crosslinking, nuclei are isolated and then nuclear extracts are prepared. Formaldehyde-fixed cells are highly resistant to restriction enzyme digestion or DNase I treatment. Therefore, soluble chromatin can be produced efficiently only by mechanical shearing [10, 11]. Sonication is a rapid and simple way to shear chromatin fragments and to generate rather uniformly sized pieces of DNA with well defined lengths. The addition of glass microbeads prior to sonication improves the shearing efficiency [18]. The conditions for sonication with a particular sonicator have to be b}

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Fig. 7.2 Examples of preliminary setup experiments before ChIP cloning. (a) Effect of sonica-tion conditions. DNA fragments, the majority of which were between 500 and 1600 bp long, were generated with four 20-s pulses from a W-250-classic sonicator (Branson) equipped with a microtip and operated at setting cycle 50%, output control 5. Between each pulse the samples were allowed to cool for 20 s in an ice bath. DNA was purified and analyzed by agarose gel electro phoresis and ethidium bromide staining. (b) HNF4a ChIP experiment in Caco2 cells for HNF1 a as positive target. A ChIP experiment was performed in Caco2 cells with an antibody against HNF4a (IPP HNF4a) orno antibody (noAB). After DNA purification, samples were subjected to PCR with primers designed for the HNF1 a promoter as the HNF4a-positive target. A mock probe and a portion of the total input sample were also examined by PCR.

tested beforehand, because the sonication time and number of pulses required vary, depending on the sonicator, cell type, and extent of crosslinking. Therefore, DNA is isolated after fragmentation and analyzed by agarose gel electrophoresis and ethidium bromide staining to determine how many cycles of sonication are needed to shear the chromatin to a certain size range. An example of such an experiment for the human epithelial cell line Caco2 is shown in Figure 7.2a.

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