GSM8944965: fibrosis livers, thioacetamide treated-3; Mus musculus; RNA-Seq
Frozen mouse liver tissues from control, TAA-induced cirrhosis, and regression groups (n = 3 per group) were used for single-cell fixed RNA sequencing. Tissue dissociation and fixation were performed using the Chromium Next GEM RNA Profiling Sample Fixation Kit (10x Genomics, PN-1000414, CA, USA) following the manufacturer's instructions. Briefly, tissues were weighed to determine the required volume of Fixation Buffer (1 mL per 25 mg of tissue), placed on a pre-chilled glass dish on dry ice, finely minced, and incubated in Fixation Buffer at 4°C for 16–24 hours. After centrifugation at 850g for 5 minutes at room temperature (RT), the supernatant was carefully removed. The pellet was washed twice with chilled PBS and resuspended in Resuspension Buffer on ice. Dissociation Solution (10x Genomics) was pre-warmed to 37°C for 10 minutes, and 2 mL was added to each sample. Samples were transferred into Miltenyi C tubes (Miltenyi Biotec, Cat#130-093-237) and processed using a GentleMACS Octo Dissociator with Heater (Cat#130-096-427) according to the recommended protocol. The resulting cell suspensions were filtered through MACS SmartStrainers (30 μm, Cat#130-098-458), and viable cells were counted using trypan blue or propidium iodide staining (Sigma, Cat#P4170). Finally, single-cell suspensions were stored at –80°C in Quenching Buffer supplemented with Enhancer and Glycerol. Gene expression profiling was conducted using the Chromium Fixed RNA Profiling Reagent Kit for Singleplexed Samples (10x Genomics, CG000477 Rev C), employing the Mouse Transcriptome Probe Set. Approximately 2,000,000–3,000,000 fixed cells were hybridized for 18 hours at 42 °C with whole-transcriptome probe pairs. After post-hybridization washes, cells were encapsulated in gel beads-in-emulsion (GEMs) using the Chromium X system and Chip Q. Within each GEM, ligated probe pairs were barcoded with 10x GEM Barcodes and Unique Molecular Identifiers (UMIs), followed by heat denaturation and recovery. The barcoded products underwent pre-amplification and purification using SPRIselect beads. Final libraries were generated via sample index PCR using the Dual Index Kit TS Set A and were further purified by size selection. Library quality was assessed using an Agilent Bioanalyzer, and concentrations were determined using the KAPA Library Quantification Kit (Roche). Libraries were sequenced on the DNBSEQ-G400RS platform (MGI Tech) in paired-end mode, using 28 bp for Read 1 (containing the 16 bp GEM barcode and 12 bp UMI) and 100 bp for Read 2 (capturing the probe insert).