I’m working on a set of ChIP-Seq samples and I’ve started to use DiffBind to proceed to my differential analysis.
My experimental design is the following :
2 conditions : Glucose (control condition) and Lactose (Induction condition)
3 epigenetic marks : H3K4me3, H3K9me3 and H3K27me3
3 replicates for each mark and each condition
2 inputs for each condition
1 mock for each condition
The three replicates for each condition are biological replicates from which I IPed my three marks :
Glucose condition :
Biological replicate 1 : H3K4me3 (1) H3K9me3 (1) H3K27me3 (1) Input (1)
Biological replicate 2 : H3K4me3 (2) H3K9me3 (2) H3K273 (2) Input (2) Mock
Biological replicate 3 : H3K4me3 (3) H3K9me3 (3) H3K273 (3)
And same for lactose condition.
Firstly, I would like to do a differential analysis between glucose vs lactose.
I ran three independant DiffBind analyses, one for each mark, where I used factor=epigenetic mark, condition=glucose or lactose and I didn’t use the treatment column. It seemed the right way to go but I’ve been wondering how DiffBind treats its variables « treatment », « condition » and «factor» under the hood. In my case, I’m not 100% sure which one correspond to my design. Do you have any insight on that matter ?
On the other hand, I also ran a single analysis including all my three marks in the factor column and condition=glucose or lactose, leaving empty the treatment column. I got very low FRiP scores comparing to the independant analyses so I didn’t go further but why such a difference ?
Also, I’m not sure how to use my inputs here. Should I put my IP samples in the glucose condition as bamControl for the differential analysis or my input samples ?
Same thing with my mock sample, how can I use it ?
Thank you for your help !