GSM8602645: 3DCS_4214; Homo sapiens; RNA-Seq
3D hydrogel culture wells were washed with PBS with recovery of the hydrogel and cells by gentle pipetting in PBS buffer, then centrifuged for 5 minutes at 750 x g, then the hydrogel/PBMC pellet was resuspended in TrypLE solution and incubated at 37°C for 10 minutes. RPMI media with 10% FBS was added and centrifuged for 5 minutes at 750 x g. The resultant pellet was washed twice with PBS to remove hydrogel matrix debris. PBMCs were resuspended in PBS and passed through a 40 µM filter, pelleted by centrifugation at 300 x g for 5 minutes, and resuspended in RPMI media with 10% FBS. FACS was then used to isolate cells from any remaining hydrogel debris and recovered cells were concentrated to 1,000 cells/µl in RPMI media with 10% FBS for downstream scRNA-seq. Single-cell RNA-seq library preparation was performed using the 10x Genomics Chromium platform with the Single Cell 3' Library and Gel bead kit v3.1 following the manufacturer's user guide.