title: GSM9251567: CD45+cells, PBMC, CD8 T cell-depleted PBMC-transferred; Homo sapiens; RNA-Seq
library_construction_protocol: The peripheral blood and salivary gland tissues were collected from immunodeficient mice transplanted with either human PBMCs or PBMCs depleted of CD8 T cells. Peripheral blood was lysed using HLB solution to remove red blood cells and obtain cell suspensions of PBMC. The salivary gland tissues were transferred to GentleMACS™ C tube (Miltenyi Biotec) containing 2 mL digestion buffer supplemented with 10% fetal bovine serum (FBS), 0.5–1 mg/mL Collagenase, and 0.1 mg/mL DNase I. Tissues were mechanically dissociated into small fragments using the GentleMACS™ Octo dissociator (Miltenyi Biotec). Samples were then incubated at 37°C for 45 min with continuous rotation to enhance enzymatic digestion. After incubation, tissue suspensions were allowed to further dissociate using the GentleMACS™ Octo dissociator. The resulting suspensions were diluted with RPMI, transferred to 15 mL conical tubes, and centrifuged. After discarding the supernatant, cell pellets were resuspended in 5 mL 40% Percoll. A density gradient was created with 2 mL 75% Percoll gently layered beneath the 40% Percoll solution. The gradient was centrifuged at 700–800 × g for 20 min at 20°C. The mononuclear cells in the middle layer were carefully collected. Finally, the cells were resuspended in fluorescence-activated cell sorting (FACS) buffer and used cell sorting. Human CD45+ cells were sorted from the pooled cells of four mice and approximately 13,500 cells with >95% survival rate were loaded into a Chromium Chip K to generate single cell emulsion using Chromium Next GEM Single Cell 5' Kit v2 and a Chromium Controller (10x Genomics). RNA-seq libraries were then prepared using Chromium Next GEM Single Cell 5' Kit v2 following the manufacturer's instructions (10x Genomics). The generated single-cell RNA sequencing libraries were sequenced using a NovaSeq 6000 platform (Illumina).