CITEseq of Human: Villous
Villous leukocytes (N=10) were thawed, washed, filtered, and stained with Ghost Violet 450  (Tonbo Biosciences, San Diego, CA) for 30 min in the dark at 4C. Samples were washed with a cell staining buffer (1X PBS with 0.5% BSA), Fc blocked for 10 min (Human TruStain FcX, Biolegend, San Diego, CA), incubated with a cocktail containing CD14 (FITC) and CCR2 (APC) and 0.5 g each of oligo tagged CD9 (TotalSeqTM-B0579), FOLR2 (TotalSeqTM-B0427), HLA-DR (TotalSeqTM-B0159) and CD62L(TotalSeqTM-B0147) (Biolegend, San Diego, CA), for 30 min at 4C. Samples were washed four times with 1X PBS , filtered using Flowmi 1000 L pipette strainers (SP Bel-Art, Wayne, NJ), and resuspended in 300 L FACS buffer.  CD14+CCR2+/- cells were sorted on the BD FACS Aria Fusion, counted in triplicates on a TC20 Automated Cell Counter (BioRad, Hercules, CA), washed, and resuspended in PBS with 0.04% BSA in a final concentration of 1500 cells/L. Single-cell suspensions were immediately loaded on the 10x Genomics Chromium Controller with a loading target of 20,000 cells. Libraries were generated using the 3 (V3.1) and Feature Barcode Library Kit per manufacturers instructions (10x Genomics, Pleasanton CA). Libraries were sequenced on Illumina NovaSeq with a sequencing target of 30,000 gene expression reads and 10,000 feature barcode reads per cell.