Cross-linking protein-DNA in ChIP-exo

A cross-link is a bond that links one polymer chain to another. These bonds can be formed between two proteins, two regions of a DNA strand or between proteins and DNA, as it is the case in ChIP-exo.

There are several agents that can form cross-links but formaldehyde is the one used in ChIP-exo. Formaldehyde induces protein-DNA and protein-protein crosslinks and is a common reagent for molecular biology experiments. One of the advantages of using formaldehyde is that the crosslinks may be reversed by incubating the sample at 65-70 °C.

After adding formaldehyde, the sample needs to be incubated at room temperature for 25 minutes in order to form the cross-links. Since formaldehyde can be toxic to cells if exposed for long periods of time, it needs to be removed from the solution. To do so, the sample should be centrifuged at 6500 x g for 5 minutes at 4°C in order to avoid further metabolic activity. Once the centrifugation is done, the supernatant should be discarded and the pellet resuspended in 50 mL ice-cold Tris buffer saline (TBS), vortexed and centrifuged again using the same settings. This step should be done 3 times, transferring the resuspended pellet after the last centrifugation to an Eppendorf tube, and the final volume should be 1 mL.