I have a question about pooling libraries for RNAseq. I'll really appreciate it if you could comment on this.
I have 12 samples that I would like to run RNAseq with. The 12 samples were collected from 2 different fungal cultures under 2 different conditions, and 3 replications under each condition.
samples 1-3 fungus 1 condition 1
samples 4-6 fungus 2 condition 1
samples 7-9 fungus 1 condition 2
samples 10-12 fungus 2 condition 2
Samples 1-6 were extracted from pure fungal cultures under condition 1, however, samples 7-12 were extracted from fungal cultures under condition 2 and the fungal cultures were contaminated with plant tissue, bacteria, and protozoa. I am specifically interested in the gene expression of the fungal tissue. And my goal is to compare gene expression between fungus 1 and 2, and condition 1 and 2.
I was wondering, because of the contamination in samples 7-12, is it possible to unequally pool the libraries when combining all the libraries? ( increasing the amount of RNA libraries of samples 7-12 to increase the sequencing depth of the fungal RNA in the samples). Would that be acceptable? Would it give me any trouble when analyzing the data?
Thank you for your time and help!