FAIRE in human cells is illustrated on the left, while preparation of the reference is illustrated on the right. For FAIRE, formaldehyde is added directly to cultured cells. The crosslinked chromatin is then sheared by sonication and phenol-chloroform extracted. Crosslinking between histones and DNA (or between one histone and another) is likely to dominate the chromatin crosslinking profile (Brutlag et al. 1969; Solomon and Varshavsky 1985; Polach and Widom 1995). Covalently linked protein–DNA complexes are sequestered to the organic phase, leaving only protein-free DNA fragments in the aqueous phase. For the hybridization reference, the same procedure is performed on a portion of the cells that had not been fixed with formaldehyde, a procedure identical to a traditional phenol-chloroform extraction. DNA resulting from each procedure is then labeled with a fluorescent dye, mixed, and comparatively hybridized to DNA microarrays. In this case, we used high-density oligonucleotide arrays that tile across the ENCODE regions of the human genome (30 Mb).
