RNA-Seq of human platelets in Triple Negative PMF patients
Platelets Rich Plasma (PRP) was collected from fresh PB. PRP was diluted with an equal volume of platelet washing buffer supplemented with Apyrase (0.05U/ml), Hirudin (0.08U/ml) and Prostaglandin E1 (PGE1 0.3M). Diluted PRP was centrifuged at 450g for 5 to separate platelets (PLTs) from plasma. PLTs pellet was resuspended with 1.5ml PWB and filtered through a 5m filter to remove residual WBC. PLTs purification was based on the MACS Cell Separation technology to remove any residual WBC and RBC. PLTs were incubated with appropriate amounts of anti-CD235a and anti-CD45 antibodies and column-purified. PLTs were then lysed in 700l QIAzol Lysis Reagent and total RNA was extracted using the miRNeasy Micro Kit following manufacturers instructions.
RNASeq_Plt_001