I have 60 birds in a study, we split up the birds so that there are 15 birds per treatment (so 4 treatments in total), and at 3 different time points, 5 birds were sacrificed Each bird contributed 6 different sample types(different areas of the body were sampled). I want to study the influence of treatments (ie. contrast different treatments), but I already know that both sample type and time point a major impact on gene expression expression.
For example, I want to know at timepoint 0, sample type 1, which genes are deferentially expressed between treatment 1 and treatment 2, or between treatment 2 and treatment 4, etc.
Should I split the data up into 18 smaller datasets so that each smaller dataset only contains samples of the same sample type and time point, then use design= ~treatment?
Alterantively would design=~Site+Timepoint+Treatment on my entire dataset tell me give me a good idea of genes that are differentially expressed after accounting for site and timepoint? Are there interaction terms I need to take into account? Thank you!