GSM9513606: nonPMC, Rep6, Day2; Homo sapiens; RNA-Seq
Monocyte isolation: Fresh whole blood was obtained from an ongoing study at the University of Rochester Medical Center (n=8). Blood collected in 8 mL ACD tubes was layered over Ficoll Histopaque and centrifuged at 2000 RPM for 25 minutes (4/3 brake). The plasma layer was partially removed, and the PBMC ring was collected into a 50 mL conical tube and centrifuged at 500 ×g for 5 minutes (9/9 brake). Monocytes were isolated using CD14+ MicroBeads following the manufacturer's instructions, resuspended, and cultured in 10% RPMI (with 1% PSG) for downstream assays. Platelet isolation: Fresh whole blood in 8 mL ACD tubes was centrifuged at 250 ×g for 15 minutes (2/2 brake). The plasma layer was gently transferred to a 50 mL conical tube, PGI2 was added to 1 μg/mL, and samples were centrifuged at 1000 ×g for 10 minutes (5/5 brake). Plasma was removed, and platelet pellets were slowly resuspended in Tyrode's solution supplemented with 10% ACD (v/v) and 1 μg/mL PGI2. Platelets were washed by repeating the centrifugation step and resuspended in 1.5 mL Tyrode's solution. Platelets were activated with 1 μg/mL collagen for 20 minutes at 37◦C prior to coculture with monocytes.Platelet-monocyte co-cultures and RNA extraction:Monocytes were cultured alone or with autologous platelets at a monocyte-to-platelet ratio of  1:100, at a final concentration of 0.3 ×10^6 monocytes/mL in RPMI supplemented with 10% FBS. Monocytes were collected at 24 h and 6 days. Cells were centrifuged at 500 ×g for 5 minutes, the supernatant was removed, and pellets were resuspended in 80 μL MACS buffer (0.5% BSA + 2 mM EDTA in 1X DPBS) with 20 μL Anti–HLA-DR MicroBeads. Samples were incubated for 20 minutes at 4◦C in the dark, washed with 1 mL MACS buffer by centrifugation (500 ×g, 5 minutes, 9/9 brake), and resuspended in 500 μL MACS buffer. Monocytes were positively selected using MS columns on a MACS Manual Separator according to the manufacturer's protocol. Recovered cells were pelleted and lysed in 500 μL TRIzol™. Chloroform (100 μL) was added, mixed, and incubated for 5 minutes at room temperature. Lysates were centrifuged at 12,000 ×g for 15 minutes at 4◦C, and the aqueous phase was collected. An equal volume of 100% ethanol was added, and RNA was purified using the Qiagen RNeasy Mini Kit per the manufacturer's instructions. Libraries were prepared using the Illumina Stranded mRNA Library Prep Kit and sequenced as paired-end 150 bp reads on a NovaSeq X 10B flow cell.