Hello, I am running a glm analysis with edgeR on 64 biologically independent samples, with 3 factors. I have sex (M and F), age (P0, P7, P15, P30), and genotype (Control, Knock-Out). I have two separate questions, 1) How does genotype effect males and females differently (at each timepoint) 2) Within each genotype (i.e. males only) what are the differences between genotypes over time. I began with a csv file that provides relevant information for each sample and followed the User guide's suggestion of pasting each factor together to form groups.

>Genotype<-Samples$genotype
>Stage<- Samples$stage
>Sex<-Samples$sex
>Group <- factor(paste(Genotype,Stage, Sex,sep="." ))

After this, I am not sure how to create my design matrix to ask the specific questions I am interested in?

You do just like it says in the edgeR User's Guide

design <- model.matrix(~0+Group)

Which will now compute the average for each group (e.g., Control.P0.M, Knock-Out.P0.F, etc) and you can now make any comparisons you want using makeContrasts. But do note that you should probably convert Knock-Out to KO or KnockOut or whatever, because the '-' in KnockOut will be interpreted as a minus by makeContrasts. Presuming you had already done so, you could compute the difference between male and female controls at P0 by doing

makeContrasts(Control.P0.M - Control.P0.F, levels = design)

And similarly for any other contrast you might care to make.