GSM9669311: MASH, VA-siAREL1-LNP treated-1, scRNA; Mus musculus; RNA-Seq
Frozen liver tissue samples were sectioned into fragments smaller than 0.5 cm on ice. Tissue fragments were homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938) in 2 mL of ice-cold Nuclei EZ lysis buffer (Sigma, cat. no. NUC-101) on ice, with 25 strokes of pestle A (loose) followed by 25 strokes of pestle B (tight). After homogenization, 3 mL of additional ice-cold lysis buffer was added, and the homogenate was incubated on ice for 5 minutes. The homogenate was centrifuged at 500 × g for 5 minutes at 4°C to pellet the nuclei. The supernatant was discarded, and the pellet was washed with 5 mL of ice-cold lysis buffer, followed by another 5-minute incubation on ice. After a final centrifugation step, the nuclei pellet was resuspended in 5 mL of ice-cold nuclei suspension buffer (NSB; 1× PBS, 0.01% BSA, 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). For flow cytometric purification, all steps were performed under RNase-free conditions with samples maintained on ice. Nuclei suspensions were filtered through a 70 μm RNase-free strainer, stained with 0.5 μg/mL DAPI for 5 minutes on ice, and sorted on a BD FACS Aria III sorter (BD Biosciences) using a 70 μm nozzle at 20 psi sheath pressure. The instrument was pre-calibrated with BD CS&T beads, with sample and collection chambers maintained at 4°C. A sequential gating strategy was used to eliminate background and enrich intact single nuclei: (1) FSC-A/SSC-A gating to exclude low-scatter debris; (2) doublet discrimination via FSC-H/FSC-W and SSC-H/SSC-W; (3) DAPI-positive gating to isolate intact nuclei (both 2n and 4n populations). Nuclei were sorted in purity mode into pre-chilled RNase-free wash buffer supplemented with RNase inhibitor, with ≥50,000 single nuclei collected per sample. Post-sort nuclei integrity was validated via microscopy. The purified nuclei were resuspended in 2 mL of NSB, filtered through a 35 μm strainer (Corning-Falcon, cat. no. 352235), and counted. A final concentration of 1,000 nuclei/μL was used for loading onto the DNBelab C4 channel. Single-nucleus RNA-seq libraries were prepared using the DNBelab C4 scRNA Preparation Kit (MGI) following the manufacturer's protocol. Briefly, purified nuclei were diluted to a concentration of 1,000 nuclei/μL and loaded into the DNBelab C4 microfluidic chip. Barcoded mRNA capture beads and droplet-generation oil were sequentially introduced, and droplets were generated using the DNBelab C4 system, finally forming oil-water emulsion. The oil-water emulsion was gently transferred to a 0.2 mL PCR tube within 20 minutes, and 100 μL of cover oil was added to the surface. Beads capturing mRNA were recovered for reverse transcription. The mixture was then thermal cycled as follows: 42°C for 90 minutes, 10 cycles of 50°C for 2 minutes and 42°C for 2 minutes, and finally 85°C for 5 minutes. After reverse transcription, the emulsion was broken, and the collected cDNA was purified with DNA Clean Beads. Then the cDNA amplification reaction system was prepared and thermal cycled as follows: 95°C for 3 minutes, 15 cycles of 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 3 minutes, and finally 72°C for 5 minutes. Amplified cDNA was purified using 60 μL of DNA Clean Beads. Finally, indexed sequencing libraries were constructed using the reagents in the DNBelab C Series High-Throughput Single-Cell RNA Library Preparation Kit V3.0 following the steps: (1) cDNA fragmentation; (2) end repair and A-tailing; (3) adapter ligation; (4) post-ligation purification with DNA Clean Beads; (5) sample index PCR and size selection with DNA Clean Beads. The barcode sequencing libraries were quantified by Qubit dsDNA kit (Invitrogen, cat. no. Q32854). All libraries were further prepared based on the DNBSEQ-T7RS sequencing platform. The DNA nanoballs (DNBs) were loaded into patterned nanoarrays and sequenced on the DNBSEQ-T7RS sequencer using the following read length: 150 bp paired-end reads (PE150).