Single-cell RNA-seq of peripheral blood mononuclear cells from kidney-transplant recipients with stable renal function versus graft dysfunction

Kidney transplantation restores renal function in patients with end-stage renal disease, yet long-term graft failure remains common and is often driven by immune dysregulation. We performed droplet-based single-cell RNA sequencing (10x Genomics 3′ v3 chemistry) on peripheral-blood mononuclear cells (PBMCs) collected from adult kidney-transplant recipients who either maintained stable renal function (Ctrl, n = 10) or displayed biopsy-proven graft dysfunction (PTRI, n = 10). After excluding those with active infection or recent hospitalization, ten patients with stable estimated glomerular filtration rate (eGFR ≥ 60 mL min⁻¹ 1.73 m⁻²) formed the control group, whereas ten patients with at least a 25 % eGFR decline and biopsy-confirmed chronic antibody-mediated rejection constituted the dysfunction group. Approximately 10 mL of peripheral blood was drawn into EDTA tubes 6–12 months post-transplant and processed within two hours; PBMCs were isolated by Ficoll density centrifugation, washed in PBS + 0.04 % BSA, and confirmed to have >90 % viability. Equal cell numbers from donors belonging to the same clinical group were pooled to create two composite suspensions that were loaded onto a Chromium Controller for single-cell capture, cDNA amplification and library construction. Paired-end 150-bp sequencing was performed on an Illumina NovaSeq 6000 targeting ~50 000 read pairs per cell. Raw BCL files were demultiplexed and aligned to GRCh38 using Cell Ranger 3.1; barcodes with <200 or >6000 detected genes, mitochondrial reads >15 %, or predicted doublets were removed.