Hi,

Sorry I am designing an RNA-seq experiment so I need help to be wise enough before invest any money; could I please ask you if the design gives what I want? You please imagine I have 1- 3 replications of fibroblast surrounding a tumor, 2- an 3 replications organoid (3D model) of this tumor and 3 - I have 3 replications of co-cultured fibroblast with organoid; My ultimate goal is getting genes coming from the interaction of fibroblast and organoid to know which genes are reacting in an environment contains of fibroblast and tumor, so I am going to use edgeR to for differential expression the GROUP would be co-culture vs fibroblast + organoid; Do you think this is right?

    > group
                   group batch
    fibroblast1        fb        1
    fibroblast2        fb        1
    fibroblast3        fb        1
    organoid1          or        2
    organoid2          or        2
    organoid3          or        2    
    co-cultured1       co        3
    co-cultured2       co        3
    co-cultured3       co        3

    >

group= as.factor(c(rep ("co",3), rep("fb+or", 6)))

y <- DGEList(counts = counts, group = ~group + batch)

You are planning this experiment? You haven't done anything yet? Do not prep all your samples on three different days. Prepping all samples of one type on one day is the worst thing you could possibly do, it will make your results almost meaningless.

The best thing to do would be to do all the RNA extractions on the same day, and do all the library preps on the same day, so you have no batch effect. You can have Jill do all the RNA preps on Monday, and Henry do all the library preps on Wednesday, but do not have Sarah do the co-culture ones while Aaron does the fibroblasts and Christine does the organoid samples, or have Sarah do the fibroid preps one day, and the co-cultures a different day, and the organoids on a third day. (Running the samples on separate lanes or runs on an Illumina instrument is perfectly fine)

If you cannot do all the preps on the same day, absolutely do not do all the sample of one type on one day. An arrangement like the one below would be far better than what you outlined

fibroblast1        fb        1
fibroblast2        fb        2
fibroblast3        fb        3
organoid1          or        1
organoid2          or        2
organoid3          or        3    
co-cultured1       co        1
co-cultured2       co        2
co-cultured3       co        3

And of course, 3 replicates is the absolute bare minimum...you'd be much happier with, say, 5. With three, if two cluster closely, and one is a bit away, it's hard to say if that's normal variation or an outlier you can easily justify omitting. But if 4 cluster together, and only one is out on its own, it's easier to justify removing it.