Reversal of cross-linking in ChIP-seq

Question

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. A summary of the protocol for ChIP is here. Briefly, the steps are:

1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked;
2. The DNA-protein complexes are sheared into ~500 bp DNA fragments by sonication or nuclease digestion;
3. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody;
4. The associated DNA fragments are purified and their sequence is determined.

After step 3 (immunoprecipitation of the protein-DNA complex), it is my understanding that the protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K.

Can anyone tell me if during this step the DNA at site of cross-linking is included in further sequencing step or is washed away with the protein itself?

Answer 1

After the crosslink reversal step, you still have some DNA connected to protein fragments.

So at that step, instead of doing a normal DNA purification, we also need to use a phenol-chloroform extraction or special columns to seperate DNA from protein fragments.

Thermofisher usually gives really good explanations too.

Please let me know if I didn't understand the question correctly! :)