GSM5725840: Plate13and14; Homo sapiens; OTHER
Blood from enrollees was centrifuged at 400 x g for 10 minutes at RT to remove platelet rich plasma. PBMCs were isolated by Ficoll-Paque PLUS gradient centrifugation using SepMate-50 tubes following the manufacturer's protocol. PBMCs were cryopreserved in freezing solution (90% FBS/10% DMSO). Once all the samples were collected, PBMCs were thawed and washed with RPMI. Library was prepared per BD's protocol. In brief, the reagents for PCR1 including the BD Human Immune Response Panel and a custom panel of 488 genes were added to the beads. Samples were aliquoted into  PCR tubes and incubated for 10 cycles according to BD's protocol for PCR1. A double size selection was performed to remove high genomic DNA fragments. AMPure XP SPRI beads to the PCR products. After incubation, the supernatant is recovered followed by purifying the supernatant withan additional AMPure XP beads (sample tags and antibodies). The RNA tube was washed twice with 80 % ethanol. 550 μl of supernatant were removed from the antibody tube followed by two washes with 500 μL of 80 % ethanol. The cDNA was eluted off the beads using 30 μL of BD elution buffer and then transferred to a 1.5 mL tube.