Experiment design

It is a bit not traditional in my book

In the case of RNA-Seq for differential expression analysis, simply speaking you ought to have at least 2 technical replications for baseline/control because the available packages are not able to compare single sample to a single sample.

Extra information

Consider technical replication, biological replication and individual replication. In a traditional sense and little do people make the connection, N should indicate individual replication but biological replication or technical replication if what you want to control is the inter-experimental error.

Yellow = technical replication Blue = Biological replication Different occasion to actually perform the experiment (3 different arrow to the same green) = individual replication So in this case the N number would be 3 (instead of 9)

Yellow = technical replication Blue = Biological replication Same occasion to perform the experiment (1 same arrow to the same green) = individual replication So in this case the N number would be 1 (instead of 3 or 9)

But this is just an idea. For example, what if I, for the sake of sequencing cost, to run all 9 samples on 3 lanes using 3 indexes in the same sequencing run? I would say that depends on how you see it. I would see that as a better approach to control for the technical error of the sequencer cause that should never be the concern of the biologist in the ideal case. Anyway that is up to you (it is not a concern when you publish your NGS data at the time of writing) but I still prefer to have completely separated biological replications connected by controls.