title: GSM9330223: 004K-2W_visium kidney xenograft; Macaca fascicularis; Sus scrofa; OTHER
library_construction_protocol: Kidney xenograft tissues were collected from pig-to-primate transplantation models at designated timepoints. Tissues were immediately snap-frozen in liquid nitrogen and stored at -80°C until processing. Total RNA was extracted using TRIzol reagent (Invitrogen) or RNeasy Mini Kit (Qiagen) following manufacturer's protocols. RNA quality was assessed using Agilent 2100 Bioanalyzer with RIN > 7.0 required for sequencing. *library construction protocol (Bulk RNA-seq) Bulk RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit. Briefly, poly(A) mRNA was purified from total RNA using oligo-dT magnetic beads, followed by fragmentation, first-strand and second-strand cDNA synthesis, end repair, A-tailing, adapter ligation, and PCR amplification. Libraries were quantified by Qubit and quality-checked on Bioanalyzer before sequencing. *library construction protocol (snRNA-seq) Single-nucleus RNA-seq libraries were prepared using the 10x Genomics Chromium Single Cell 3' v3.1, v4 kit. Kidney tissues were dissociated to isolate nuclei using a Dounce homogenizer in nuclei isolation buffer. Nuclei were filtered, counted, and loaded onto the Chromium Controller to generate single-nucleus GEMs. Libraries were prepared following the manufacturer's protocol including cDNA synthesis, amplification, and library construction. *library construction protocol (Visium) Visium spatial gene expression libraries were prepared using the 10x Genomics Visium Spatial Gene Expression kit. Fresh-frozen kidney tissue sections (10 μm) were placed on Visium spatial gene expression slides, fixed, stained with H&E, and imaged. Tissue was permeabilized to release mRNA which bound to spatially-barcoded oligonucleotides on the slide. Reverse transcription, second strand synthesis, and library preparation were performed following the manufacturer's protocol.