Hi everyone! I am very new to the RNA-seq analysis and having quite a dilemma to design my experiment for the RNA-seq, in terms of the number of replicates required for each group of sample, with the consideration of budget constraint.

So here is the design of my experiment:
I am deriving different models of stem-like cells  (or cancer stem cells, CSC) derived from HepG2 cell line, with a total of 3 models. So for each model, HepG2 cells were treated/cultured in different culture condition to enrich CSCs, meaning at the end of the day the total sample is 4 (1 parental HepG2 cells, 3 CSC-enriched models derived from HepG2 cells). 

My questions here, if I do three replicates of these treatment/models, are they considered to be biological replicates? How are these considered biological replicate since they all come from the same cell line, just different treatment for the treated samples, aren't these technical replicates? Because I have read that technical replicates are unnecessary for RNA-seq but my concern is that whether the data will be reliable and publishable. But then again, is it right to do triplicates for RNA-seq from these samples? Plus, another concern are also considering the budgetary constraint of sequencing 12 samples, which are not affordable on our part.

Any thoughts? Sorry if my write-up is confusing, will really appreciate inputs from experts on this matter! Thanks!!!
You're correct that technical replicates are not necessary for RNA-seq, however biological replicates are always necessary for statistical analyses. Without replicates you cannot have any p-values.

A technical replicate in RNA-seq, in my experience, refers to taking one tube of RNA and then making multiple sequencing libraries from that tube. This is to control for errors in pipetting/PCR/anything technical during the library prep procedure. The kits available nowadays are so reproducible that this really isn't necessary. Biological replicates, however, are needed.

If I were designing the experiment, I would take the parental HepG2 cells, split them into three separate plates, then when the plates reach the appropriate confluence, I would treat them to generate the CSC-enriched models. Although there is an original single plate of cells, each of these biological replicates have undergone the CSC-enrichment separately, therefore any differences should be representative of this CSC-enrichment process (or biological differences).

You ***COULD*** get away with doing duplicates if sequencing 12 samples is absolutely unaffordable, some programs such as DESeq2 and edgeR are able to perform analyses with fewer replicates, but it really is not advisable. You can pool your 12 samples and run them down a single sequencing lane to save money, you'll have lower depth but you can always resequence and pool the raw fastq files if necessary.

I hope all of this is clear, please let me know if you have further questions.