Ai_heart_MG_scRNAseq
Heart tissues were digested in 1.5 mL centrifuge tubes containing 1 mL of 2 mg/mL type II collagenase dissolved in 3  PBS. After 30 minutes of dissociation on a shaker at room temperature, cell suspensions were filtered through a 40 m nylon strainer. The suspension was then washed, centrifuged (600 g for 5 min), and resuspended in 3  PBS to remove debris. The final cell suspension, with >90% viability (Trypan blue staining) and a concentration of ~1000 cells/L, was prepared for library construction. The entire process was performed on ice. Beads with unique molecular identifiers (UMIs) and cell barcodes were loaded to near saturation in Gel Beads-in-Emulsion (GEMs), ensuring each cell was linked to a bead. After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. The beads were then collected for reverse transcription, during which each cDNA molecule was labeled with a UMI and cell barcode. cDNA synthesis, second-strand synthesis, adaptor ligation, and amplification followed. Sequencing libraries were prepared using whole-transcriptome amplification to target the 3' end of transcripts linked to the cell barcode and UMI. Library construction followed the standard Chromium Single Cell 3' (v3.1) protocol. Library quantification was done using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). Sequencing was performed on a NovaSeq6000 (Illumina) with 150 bp paired-end reads.