I have been performing RNA-seq analyses using DESeq2 a particular way and I just wanted to verify whether my logic is sound.
Basically my experimental condition is such - I have a cell line in which there is a heterozygous mutation at a gene of interest. This Heterozygous mutation (amongst other things) leads to constitutive activation version of the gene of interest. I wanted to do an RNA-seq analyses to compare the transcriptome of this cell with the activating mutation to the same WT cell line, but activated by a ligand.
Because the mutant cell line is heterozygous it still has one copy that is WT and my concern is amongst the differentially expressed genes are genes in which are upregulated by WT in the vehicle setting compared to WT with the ligand and not Mutant vs WTligand. To combat this we also performed RNA-seq on WTveh and then performed a DE analysis with WTligand. I decided (maybe incorrectly) that if it has a padj < 0.05 and log2foldchange > 1 it is considered upregulated in unliganded vs liganded in WT cells. This was the list I removed from the Mutant vs WTligand comparison after I performed the DE analysis there.
Is this an appropriate approach? Does anyone have a better suggestion? I actually did this exact thing to two different cell lines in which there is a corresponding heterozygous mutation (separately, of course)
I appreciate everyone's help,