RNA-Seq analysis and biological replicates.

Question

[tmms]

I want to analyze RNA_Seq data that I found on the internet to practice. I found 8 RNA_Seq datasets (four different immune cells from mice and everytime two biological replicates). The goal of the analysis is to discover if there are genes that are differentially expressed.

I want to used edgeR to analyze the data. In order to order to do that, I need to specify a design matrix. There I am stuck. Cell type is one factor with 4 different levels in this analysis, but what about biological replicate? Should it also get an own factor with two levels?

I think biological replicate should not get a own factor, but I can not really explain why. It is a hunch.

Thanks in advance.

Answer 1

[Matteo Schiavinato]

You might find this useful: C: DESeq2 proper design setting

Answer 2

[Carlo Yague]

If the all the replicates 1 were processed together and all the replicates 2 processed separately, then yes, you need to include that information as a factor because some variability could be explained by the level of the replicate. Otherwise, you should not include it.

Answer 3

[Thind amarinder]

Have a look on this paper .. https://rnajournal.cshlp.org/content/22/6/839.long