Illumina HiSeq 2500 paired end sequencing; Functionally-relevant splicing of resident pre-mRNAs in anucleate platelets upon activation by physiological stimuli
Functionally-relevant splicing of resident pre-mRNAs in anucleate platelets upon activation by physiological stimuli
Fifteen healthy volunteers (all males with no cardiovascular risk factors) who refrained from taking any medication were enrolled. A written informed consent was obtained from each subject. The protocol was approved by the local ethical committee of the University of Campania \"Luigi Vanvitelli\". Fifty ml of blood from each donor were collected into 0.105 M sodium citrate tubes (1:10 V/V) and processed within 15 min from sampling. For platelet isolation, blood samples were centrifuged at 160 × g for 15 min at room temperature. The platelet-rich plasma obtained was then filtered using a specific leukocyte removal system (Pure Cell PALL, Pall Italia), as described earlier13. Leukocyte contamination was assessed by flow cytometry and rtPCR analysis of leukocyte (CD-45) and platelet (CD-41) specific antigen mRNA, with GAPDH RNA used as control, as described in Supplementary Information (Methods section). Purified platelets from each donor were split in 3 aliquots: a first aliquot was immediately processed and used as control (baseline: T0), two aliquots were treated with Collagen (COLL, 60µg/mL) or Thrombin Receptor Activating Peptide (TRAP 25µM) under continuous stirring for 120 minutes at 37°C before processing. Platelet aggregation was verified in all samples before and after stimulation by the formation of macroaggregates and confirmed by light transmission aggregometry as described in Supplementary Information (Methods section). After two hours of stimulation, platelets were centrifuged at 2,000 x g at 4 °C, washed 3 times with PBS and then stored at -80°C until further processing. Each replicate/sample (CTRL,COLL and TRAP) is pool of five donors and the average age of donors is about 40 +/- 3 years old.  Purified platelets from each of the 15 donors were subjected to total RNA and protein extraction using the mirVana PARIS kit (Thermofisher scientific) according to the manufacturer's instructions. 600 µl of ice-cold Disruption Buffer from the kit were added to each platelet aliquot (the 15 donors) and 3 pools of 5 samples/each were made. One third of each pool was withdrawn for protein extraction, carried out as described13, and the remaining was processed for RNA purification as described earlier13. RNA concentration in each sample was assayed with a ND-2000c spectrophotometer (Thermo Scientific) and its quality and integrity assessed with the Agilent 2100 Bioanalyzer with Agilent RNA 6000 pico kit (Agilent Technologies). Protein concentration was determined using Bradford Protein Assay and EZQ Protein Quantitation Kit. Three independent biological replicates for each treatment and for the CTRL were used for all subsequent analyses.  Indexed libraries were prepared using 250 ng of total RNA as starting material, with TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.).
Experimental Factor: compound
Thrombin Receptor Activating Peptide
Experimental Factor: dose
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