Formaldehyde Cross-Linking

Abstract

Formaldehyde cross-linking of DNA to associated proteins is a relatively straightforward method, but it is also the most critical step in the chromatin immunoprecipitation (ChIP) and 3C analyses. Although formaldehyde is a highly permeable cross-linker, its maximum cross-linking efficiencies are estimated to be at approximately 1% for mammalian cells because reactivity is limited to amines. Therefore, a relatively large number of cells are required for 3C and ChIP-based assays. Five hundred million cross-linked diploid cells are equivalent to approximately 1.66 fmol of the genome. Thus, only approximately 100 amol of genomic copies is analyzed for one ChIP assay. Because the quality of cross-linked chromatin can vary, even when generated under near-identical conditions, it is preferable to generate multiple large batches. This protocol describes growing and cross-linking IMR90 primary human fibroblast cells for ChIP analysis. For other cell types, some modification of the protocol is necessary.