GSM8107938: Second_kidney_xeno_recipient_PBMC_scRNA-seq_6h; Homo sapiens; RNA-Seq
Fresh kidney tissues were minced rapidly in a cell culture dish followed by washing with DPBS buffer. Minced tissue was resuspended in 5mL of 37°C pre-warmed 0.25% Trypsin for 20 min with mechanical dissociation using serological pipette every 5 min. Following digestion, dissociation mix was filtered through a 100µm strainer. The remaining large tissues on the strainer were further ground and washed to collect additional cells. Cell debris and dead cells were removed using Miltenyi Biotech Dead Cell Removal Kit before loading for 10X Genomics single-cell RNA-seq (v3.1 Chemistry). Longitudinal single-cell RNA-seq of the recipient's PBMCs were prepared at the same time after collecting all samples across timepoints. Fresh PBMCs of each timepoint were first collected through gradient centrifugation, followed by cryopreservation of the prepared PBMCs according to the 10X Genomics protocol (protocol number: CG00039). Upon collecting all PBMC samples, the frozen cells were thawed at the same time, then loaded for 10X Genomics single-cell RNA-seq (v3.1 Chemistry). prepared signle cells were sequenced using 10X Genomics single-cell RNA-seq (v3.1 Chemistry) following manufacturer's protocol.