single-nucleus RNA sequencing (snRNA-Seq) of hippocampus in db/db mice following EV therapy

Hippocampi were collected from db/db mice 4 weeks post-injection with either EV or PBS, with WT mice as controls. All procedures were conducted on ice within a 4C ultra-clean workbench. Nuclei were isolated from fresh-frozen tissue using a modified protocol. Briefly, hippocampal tissue was homogenized in chilled Nuclei EZ Lysis Buffer (NUC101, Sigma-Aldrich, USA) using a glass dounce tissue grinder (FIS#K885300-0002, Kimble Kontes, USA). Homogenates were filtered through 70 um strainers, centrifuged at 500 g for 5 minutes at 4C, or alternatively, cleaned via density gradient centrifugation employing Debris Removal Solution (130-109-398, Miltenyi Biotec, DE). Resuspended nuclei underwent further centrifugation and filtration through a 35 um strainer. Approximately 6000 nuclei per sample were captured using 10x Genomics Single Cell 3' Reagents kit. Sequencing was performed on an Illumina NovaSeq 6000 with 150 bp paired-end reads by Genechem (Shanghai, China). The Cell Ranger Analysis Pipeline (v6.0.2) facilitated the generation of sequencing libraries, and resulting unique molecular identifier (UMI) count matrices were processed using Scanpy (v1.8). Quality control eliminated cells with high mitochondrial content (PMC <= 5%), low UMI counts (<= 100,000), and outside the 200-8,000 gene range. A total of 53,307 single-cell transcriptomes were included for analysis.