Are unequal pool sizes for RNA-seq acceptable?
 

Background:
I want to find differentially expressed genes between wild type (WT) and knockout (KO) mice of mixed background using RNA-seq (Illumina HiSeq, 50M reads standard at my core). There will be anywhere between 1-4 KO and 1-4 WT animals per litter.

Because it is difficult to isolate enough cells from one animal, I need to pool cells from WT littermates and KO littermates. I need 2 animals minimum, but 3 and up is preferable. My thinking is that each litter will be one biological replicate, and I plan to sequence RNA from 3-5 litters (3-5 biological replicates). Because the number of KO vs. WT animals within a litter varies, I may have more of one genotype than the other.

Question:
If I had this scenario:
Bio rep 1: Pool of 3 WT and Pool of 2 KO
Bio rep 2: Pool of 2 WT and Pool of 2 KO
Bio rep 3: Pool of 2 WT and Pool of 4 KO

...or any combination thereof, would this be a statistically poor design? Should I keep the number of animals in each pool constant for all biological replicates? Only within biological replicates?

Another consideration: Will this be representative of only one mating cage? Cost/time is limited to maybe one time on the sequencer, two chips.

In the age of single-cell RNAseq, there's little remaining reason to pool multiple samples like that.

BTW, I'm glad you're taking litter into account. A lot of people don't do that and then their results end up being do to a litter effect.

So you're saying what exactly? Pool size doesn't matter? Keep pool size equal at the minimum of 2 and the sequencing can take care of the rest?

I'm saying pooling is a last resort. Try to find someone local doing single-cell sequencing and you'll be able to both increase your N and eliminate pooling in one go.