I am planning to use exogenous chromatin as a spike in control with my actual sample from mouse to perform ChIP-seq for peak calling and differential binding analysis for histone modifications. this involves down-sampling the uniquely mapped read files to the calculated normalization factor from the spike in. This is seemingly helpful for peak calling and visualizing in genome browser. But I have not found any reference on whether it is considered in differential binding analysis. Since I am using Diffbind, I also could not find anything regarding this in the vignette. Could anyone please explain how this strategy might affect using the diffbind package? Or, does using spike-in for ChIP-seq normalization makes sense?
I greatly appreciate your time to read and answer to my question! Thank you in advance!
An example of this normalization process is given below(Image from Active motif's ChIP-seq spike in kit.)
