Question

Typically DamID results are normalized by using a ratio of the signal produced by the Dam methylase fused to the target protein (in this case lamin B1) divided by the signal produced by free Dam methylase. The results from lamin B1 DamID suggested that the genome could be divided into two main types of domains:  Lamin Associated Domains (LADs) and the regions between these LADs (inter-LADs).  In theory you would think that the same division could also be seen by ChIP-Seq using an antibody for example against lamin B1 for the ChIP.

How does ChIP (chromatin immunoprecipitation) work?

Answer 1

Chromatin imunoprecipitation (ChIP) is an immunoprecipitation technique where we can determine specific interactions between proteins and DNA in the cells. Like whether the protein is associated with genomic regions such a transcription factors on promoter regions . It also aims to study various regions of histone modifications, indicating regions of histone modifiers.

ChIP are of two types;

1. Native ChIP (NChIP) :It is mainly used for DNA targets of histone modifiers. In this type basically a step called crosslinking is not done.

2. Cross linked ChIP (XChIP) : It is used for DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly cross-linked chromatin as starting material.

Steps involved :

1. DNA and associated proteins on chromatin in living cells are crosslinked (this step is not done in NChIP).

2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by ultra sonication or nuclease digestion.

3. DNA fragments associated with the protein of interest are immunoprecipitated using an appropriate protein-specific antibody (in this case Lamin B1 antibody).

4. The associated specific DNA fragments are purified and their sequence is determined, these interactions are all in vivo.