I know this has been explained numerous times and for various scenarios (some of which I feel are more complicated than what I'm attempting to achieve here), but I struggle to understand how to properly design my analysis..
So what I have is one cell line, infected with either control or shRNA A and B targeting single gene, in the presence or absence of over-expressed gene X at day 2 and day7. So basically:
> colData.d sample conditionX knockdown shRNA replicate timepoint 1 1 ctr ntc ntc rep1 day2 2 2 ctr ntc ntc rep2 day2 3 3 ctr ntc ntc rep3 day2 4 4 ctr knockdown shRNA-A rep1 day2 5 5 ctr knockdown shRNA-A rep2 day2 6 6 ctr knockdown shRNA-B rep1 day2 7 7 ctr knockdown shRNA-B rep2 day2 8 8 geneX ntc ntc rep1 day2 9 9 geneX ntc ntc rep2 day2 10 10 geneX knockdown shRNA-A rep1 day2 11 11 geneX knockdown shRNA-A rep2 day2 12 12 geneX knockdown shRNA-B rep1 day2 13 13 geneX knockdown shRNA-B rep2 day2 14 14 ctr ntc ntc rep1 day7 15 15 ctr ntc ntc rep2 day7 16 16 ctr knockdown shRNA-A rep1 day7 17 17 ctr knockdown shRNA-A rep2 day7 18 18 ctr knockdown shRNA-B rep1 day7 19 19 ctr knockdown shRNA-B rep2 day7 20 20 geneX ntc ntc rep1 day7 21 21 geneX ntc ntc rep2 day7 22 22 geneX knockdown shRNA-A rep1 day7 23 23 geneX knockdown shRNA-A rep2 day7 24 24 geneX knockdown shRNA-B rep1 day7 25 25 geneX knockdown shRNA-B rep2 day7
What I basically would like to know is the effect of knockdown in ctr or geneX cells at different time points. So far I basically created new factor that combines conditionX with timepoint (condition) and analyzed the "knockdown" effect. But I feel it might be better to control for differences between the shRNA A and B on the knockdown effect.. however when I that with e.g. design= ~shRNA+knockdown, I get " model matrix is not full rank" error.. ok, now if I understood correctly I should try treating this as a nested scenario? Is that correct?
Thank you so much for any suggestions on how to approach this..