scRNASeq of Homo sapiens: Immune Cells
Bone marrow aspirates were diluted 1:1 in FACS buffer (PBS without magnesium or calcium, with 0.1% BSA and 2 mM EDTA). Peripheral blood samples were diluted 1:2 in FACS buffer. Mononuclear cell separation was performed by density centrifugation (Ficollpauqe, GE) with diluted bone marrow cells. Cells were manually aspirated and washed with FACS buffer. Red blood cells were lysed using RBC lysis buffer (Invitrogen) for 5 min at 4C. Within 1 hour of processing, cells were stained and sorted (Aria Fusion, BD) based on forward and side scatter, gated on CD45+ CD19- CD3+ (T cells) and CD45+ CD19- CD3- (nonB nonT cells). Droplet-based single cell RNA sequencing (scRNAseq) was performed using the 10x 5 Reagent Kits version 1, according to manufacturer instructions. 10X 5 kits for TCR amplification were used to generate libraries per manufacturer instructions. All sequencing was performed on an Illumina NovaSeq S4 sequencer with paired end 200 base pair read length and 25,000 reads per droplet for gene expression and 5,000 reads for TCR libraries.