GSM7152487: Tonsil 2, rep1, scRNAseq; Homo sapiens; RNA-Seq
Freshly resected tonsil tissue was washed in PBS, any visible signs of pus, blood, or burns were cut away and then tissue was further cut into smaller pieces before being mechanically disrupted by grinding and filtering. The filtered cell suspension was washed in 50 ml PBS, pelleted by centrifugation and resuspended in 20mL PBS and layered onto 30mL Ficoll (Cytiva) and centrifuged. The lymphocyte layer was harvested and washed with 50mL PBS before being pelleted by centrifugation and resuspended in 50mL PBS. 500 million cells were stained with Fc Block (Miltenyi Biotec) for 15 minutes, washed, and then stained with Miltenyi MACS anti-CD19 magnetic beads according to manufacturer's instructions. After staining cells were washed and B cells were depleted using Miltenyi MACS LD columns to deplete B cells according to manufacturer's instructions. , cells were incubated with live/dead cell marker (DCM; Invitrogen) and surface protein antibodies at 4 °C for 30 min, washed with PBS and resuspended in FACS buffer (PBS with 2% FBS). Cells were sorted into eppendorf tubes containg Sort Buffer (PBS with 0.2% BSA and 5mM EDTA). Cells were sorted on a FACSAria III sorter (BD Biosciences), with FACS Diva version 9 software. Library was performed according to the manufacter's instructions (single cell 5' v2 protocol, 10x Genomics)