I am trying to study the effect of TGF-beta between two genotypes WT and KO in MEF cells. To compare the two genotypes, we have knocked out (KO) the gene using the parental as WT control. Then we have stimulated the cells with either TGF-beta or just serum free control, these cells we have analysed at 1 hour and 24 hours. To compare these I have managed to create a design matrix for individual comparisons of each occasion which is each time point either stimulated or not and then just compare the DE between each time point eg. KO24hControl-WT24hControl.
my.design <- model.matrix(~ 0 + design, data=matrix)
KO1hControl KO1hTGF KO24hControl KO24hTGF WT1hControl WT1hTGF WT24hControl WT24hTGF
1 0 0 0 0 0 1 0 0
2 0 0 0 0 0 0 0 1
3 0 0 0 0 1 0 0 0
4 0 0 0 0 0 0 1 0
5 0 1 0 0 0 0 0 0
6 0 0 0 1 0 0 0 0
7 1 0 0 0 0 0 0 0
8 0 0 1 0 0 0 0 0
9 0 0 0 0 0 1 0 0
10 0 0 0 0 0 0 0 1
11 0 0 0 0 1 0 0 0
12 0 0 0 0 0 0 1 0
13 0 1 0 0 0 0 0 0
14 0 0 0 1 0 0 0 0
15 1 0 0 0 0 0 0 0
16 0 0 1 0 0 0 0 0
17 0 0 0 0 0 1 0 0
18 0 0 0 0 0 0 0 1
19 0 0 0 0 1 0 0 0
20 0 0 0 0 0 0 1 0
21 0 1 0 0 0 0 0 0
22 0 0 0 1 0 0 0 0
23 1 0 0 0 0 0 0 0
24 0 0 1 0 0 0 0 0
25 0 0 0 0 0 1 0 0
26 0 0 0 0 0 0 0 1
27 0 0 0 0 1 0 0 0
28 0 0 0 0 0 0 1 0
29 0 1 0 0 0 0 0 0
30 0 0 0 1 0 0 0 0
31 1 0 0 0 0 0 0 0
32 0 0 1 0 0 0 0 0
What I would like to do is have a comparison of the effect of TGF-beta between the genotypes. To compare this I was wondering if it would be possible to do like in the edgeR User guide 3.3. "Experiments with all combinations of multiple factors." https://bioconductor.org/packages/devel/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf If I would translate what they did to my design I would do like: KOvsWT.24h.TGF = (KO24hTGF-KO24hControl)-(WT24hTGF-WT24hControl). And this would show me the DE between WT and KO in TGF-beta stimulated cells at 24hours? Do I need to consider the time points?
I am trying to get my head wrapped around how to construct a good contrast and any input to this would matter a lot for me! Thanks a lot in advance!
