Approximately 500pg to 1.5 ng Total RNA was obtained using the Picopure RNA isolation kit with an on column DNAse treatment. Thereafter, the Nugen ovation RNAseq V2 amplification kit was used to generate ds cDNA. A library was made and sequenced on the Hiseq 4000 50bp reads. On average 38*10^6 reads were obtained per sample.

The problem that I encounter is that sometimes in one of the samples the total read count per gene body shows a big difference compared to the other samples. This phenomenon is not sample-specific.