I have what might be a simple question but I would really like to get this right and despite all my readings it is still not clear to me what the best option is.
I am using DESeq2 to analyse an RNAseq experiment. The design of the experiment is as follow (each sample is in triplicate):
- cell line A - untrt
- cell line A - trt
- cell line B - untrt
- cell line B - trt
The goal is to peform the following contrats:
- cell line A - untrt versus cell line B - untrt
- cell line A - trt versus cell line B - trt
- cell line A - untrt versus cell line A - trt
- cell line B - untrt versus cell line B - trt
My question is, in order to do things "correctly", should I create a separate DESeq object for each contrast using only the samples I will be using for each contrast, or should I have just a single object from which I do the contrasts that I need?
For your information, the difference between these cell lines is that "cell line A" is overexpressing gene X, while "cell line B" is overexpressing genes X and Y.
Any help would be very much appreciated...