Hello to everyone. I have regularized log2- transformed count data (from bulk RNAseq), generated by DESeq2 function rlog. I have technical triplicates of cells untreated and treated. I´ve been suggested to calculate the fold change between the triplicates for every gene to see easily a difference in the gene expression between untreated and treated conditions. Due to the triplicates are quite consistent between each other for all the genes, I decided to calculate the mean first and then make the ratios of the means (treated/untreated) to calculate the fold change. My questions are: 1) Does it make sense first to calculate a fold change from regularized log2- transformed count data generated by DESeq2 function rlog? 2) Is correct to calculate in this way a fold change? Thank you all so much for the replies and help!
rlog transformed data is generally not used for calculating fold changes. It's for other things, like PCA calculations. Just find a tutorial and follow that. No on is going to understand why you want to do things your own way when experts who figured all this out don't do things like that.
Do you really only have technical replicates, and not biological ones? Like, the same library prep run on three lanes? That's not going to be very informative at all.