RNA was prepared from PBMCs using the RNeasy Mini Kit (QIAGEN, Germany).  The pellets obtained from buffycoat were resuspended in the RLT buffer. We proceed with the addition of equal volume of 70% Ethanol (EtOH) to our already homogenized sample in RTL. It was spinned at ≥8000 x g (≥10,000 rpm) for 15sec. 700 µl of Buffer RW1 were added and the solution was spinned at ≥8000 x g (≥10,000 rpm) for 15sec. It was emptied carefully, and 500 μl of RPE Buffer were added and the solution was spinned ≥8000 x g (≥10,000 rpm) for 2 minutes. The column was emptied again and placed in a new tube and centrifuged at maximum power for 1 minute to dry. NEGEDIA Digital mRNA-seq research grade sequencing service 2.0 (Next Generation Diagnostic srl)