GSM6323493: P1; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (Cytiva, Cat#17-1440-02) density gradient centrifugation, subjected to RBC lysis, and frozen at -80C before transferring for storage in liquid nitrogen. On the day of the experiment, PBMCs were thawed at 37C, incubated in DNase I solution, filtered and then isolated using the EasySep Human T cell Isolation Kit (Stemcell, Cat 17951) following the manufacturer's instructions. Cells were seeded at 1x106 cells/ml in R10 media, and placed in a 37C incubator with or without stimulation. Cell stimulation was performed with 25ul/ml Immunocult Human CD3/CD28 activator reagent (Stemcell, Cat #10971) for 16 hours. Cells were filtered, washed with PBS/2% FBS/1mM EDTA, and resuspended in PBS/0.04% BSA solution for analysis Samples were further processed by the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine where single-cell suspensions were loaded onto a Chromium Single Cell Chip (10x Genomics) according to the manufacturer's instructions.