Single-cell RNA-seq of Human Immune Cells

Enriched hCD33⁺ cells from liver and spleen of hNSG-SGM3 mice were resuspended in PBS containing 0.04% BSA, the cell numbers were counted on the Contess II automated cell counter (Thermo Fisher Scientific), and ∼12,000 cells were loaded per channel on Chromium microfluidic chips (10x Genomics). Single-cell capture, barcoding, and library preparation were performed using the 10x Chromium version 2 chemistry according to the manufacturer’s protocol (10x Genomics). The quality of cDNA and libraries was checked on an Agilent 4200 TapeStation, quantified by KAPA quantitative PCR, and sequenced on a HiSeq 4000 (Illumina) to an average depth of 50,000 reads per cell. We quantified gene expression counts from raw sequencing data using Cell Ranger v2.2 with GRCh38. Datasets from liver and spleen of two independent experiments were normalized using Harmony (Korsunsky et al., 2019 (link)).