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 ∼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 ∼1.66 fmol of the genome. Thus, only ∼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.