I have a question around the meaning of "biological replicate" in the context of applying RNA-seq to compare two cell lines. Apologies if this is an overly naeve question.
We have two human cell lines, one of which was derived from the other. Both have different phenotypes, and we want to use RNA-seq to explore the genetic underpinnings of the difference.
If we generate one cDNA library for each sample, and sequence each library on two lanes of an Illumina GA flowcell, I understand we will have "technical replicates". In this scenario, we can expect very little difference between the two replicates in a sample. If we were to use something like DESeq to call differential expression, it would be inappropriate to treat our technical replicates as replicates in DESeq, since that would likely lead to a large list of DE calls that don't reflect biological differences.
So, I'd like to know if it possible within our model to have "biological replicates" with which we can use DESeq to call biologically meaningful differential expression.
So, two questions:
(1) If we grow up two sets of cells from each of our two cell lines, generate separate cDNA libraries (4 in total), and sequence them on separate lanes, would these be considered "biological replicates" in the sense that it would be appropriate to treat them as replicates within something like DESeq. I suspect not, since the fact that both replicates in a sample derive from a single cell line within a short period of time will mean that they will be very similar anyway, almost as similar as the technical replicate scenario. Perhaps we would need entirely separate cell lines to be considered biological replicates.
(2) In general, how would others address this - does it seem a better approach to go with separate cells and separate libraries, or would this entail extra effort for effectively no benefit?
Thanks for your time.