which one would be the better way for RNA-seq Experiment design

Question

[Manvendra Singh]

I have a few questions concerning RNAseq experiment.

we have 10 samples, 5 controls vs 5 Patients. We are going to run them into two lanes. I am wondering how should I index and pool them. Should I use 10 different indexes, pool them all and run in two lanes (that will makes two replicates) or to use 10 different indexes but pull 5 samples in one lane and 5 samples in the second lane that we will have in each pool as different indexes as possible, if so should I run 5 controls in one line and 5 cases in the second line or combine them i.e. 3 controls + 2 cases in one line and 2 controls + 3 cases in the second one.

I hope it is understandable what I wrote.

Thank you in advance.

Answer 1

[User 59]

10 plex them and put them down two lanes (i.e. all ten samples in BOTH lanes). This means you will not get any lane to lane biases. And if you have a lane failure, you'll still have a lane of usable data! If you have two good lanes, then you can combine the data and run it as the 5v5 comparison from your 20 outputs.

[Manvendra Singh]

Thanks Daniel, I would do it in the same way!