Differential expression analysis in DESeq2 for an experiment with multiple variable

Question

[Mozart]

Dear all, I am approaching a kinda new experiment and wondering what is the best way to construct my design, to plug into DESeq2, in order to best address my question of interest.

• First we have, 10 replicates of mouse cells from tissue X taken at two different steps of their life cycle (let's say cycle A and cycle B). These cells were taken from lab 1. Samples were paired-end sequenced.
• Second, 10 replicates of mouse cells from tissue Y taken at two different steps of their life cycle (again, cycle A and cycle B). These cells were taken from lab 2. Samples were single-end sequenced.

All of these samples are balanced and/or paired (?), meaning that each biological replicate is taken from the same mouse each time. For example, from mouse 1 were extracted cells at both cycle A and cycle B.

So, same cell types at two separate conditions but extracted from two different labs (in two different parts of the globe). I am relatively new to this kind of design formula. But, what I want to do is:

• First, find differentially expressed genes within each lab (i.e. A vs B from lab 1; A vs B from lab 2)
• Second, try to compare everything across each lab in all the possible combinations but also caring of the different above mentioned variables.

To sum up we have a four different variables

1. cycle

2. tissue

3. replicate

4. lab

Therefore I would suggest a design matrix like the following one: ~ tissue + tissue:cycle + replicate + batch + cycle

I am just throwing an idea, I am not 100% sure.

Thanks in advance