Hoping that some of the RNAseq experts in here are having some pieces of advice on normalization/proceeding of following data analysis:
The study is about how a stressor is affecting a non-model species (no reference genome/transcriptome available) in terms of differential gene expression. We did a deep sequencing to make a reference transcriptome and sequenced the samples at a "lower depth":
Reference transcriptome de novo assembled (Trinity) from reads with a sequencing depth of 300 M (PE 2x150 nt). The statistics (TrinityStats), E50N90, BUSCO analysis, Blast2Go, Detonate (comparison of 3 assemblies - chose the best one) looks good. The reference is made from a non-stressed individual of the non-model organism.
Triplicate samples of "negative stress control", "positive stress control" and the "treatment" with a sequencing depth of 25M (PE 2x75nt)
How is the best way to use the reference transcriptome in order to determine differential gene expression of the samples? any tips/tricks on how to normalize?