I have 72 samples consisting of 36 from Day 3 and 36 from Day 6, with 4 types of cell lines (A, B, C, D) and 3 conditions: treated, control, and untreated. Each sample is in triplicate.

I performed DESeq2 analysis using two different methods:

Method1

Day 3 analysis

design = ~ -1 + Condition)

Method 2

design = ~ -1 + Condition + Cellline) Day3$group <- factor(paste0(Day3$Cellline, Day3$Condition)) design(Day3) <- ~-1 + group

These two methods yield different numbers of DEGs. Could you please advise which one is correct?

Thanks

Essentially, the first method does not correct for cell line while the second one does. If cell line is a confounder that drives unwanted variation then it makes sense to include it as a covariate. Check the vignette towards PCA to diagnose that. Generally, there is usually no one "correct" answer on how to do your analysis, but hands-on guidance is unfortunately nothing we can provide here. In general, biostars.org has a broader audience for these sorts of questions. Please make sure you only include relevant code. Here the design alone would be sufficient, given that the rest would be identical between methods.