GSM8381531: Liver endo, CDAA-tdTnegtive-rep2, RNAseq; Mus musculus; RNA-Seq
After killing the mice, 30 ml of HBSS (Invitrogen, 14175103) was perfused through the portal vein, and then a portion of the liver tissue was placed in cold HBSS containing 0.4 mg/ml pronase (Roche, 10165921001) and 1 mg/ml collagenase type IV (Worthington, LS004188). The livers were then cut into small pieces and incubated in cold HBSS (Invitrogen, 14026126) containing 0.5 mg/ml pronase, 1 mg/ml collagenase type I (Gibco, 17100017), 1 mg/ml collagenase type IV and 50μg/ml DNase I (Worthington, LS002139) in 10 ml HBSS digested at 37 °C with shaking for 30 min. Liver samples were filtered through a 70 μm cell strainer. Nonsubstantial cells were then isolated by gradient centrifugation (1500 g, 20 min) between two density pads of 25% and 50% Percoll (Sigma, P4937-500ML). After isolation and centrifugation at 4 °C, 1 ml of erythrocyte lysis buffer (eBioscience, 00-4333-57) was added and centrifuged for 5 min at room temperature. To stop the reaction, 9 ml of cold PBS was added and centrifuged at 700 g for 5 min at 4 °C and the supernatant was discarded. After lysis of erythrocytes, the cells were washed twice with PBS for the next step. Dissociated hepatic nonparenchymal cells were resuspended in an appropriate amount of buffer containing 1% Fc block (Catalog No. 14-0161, eBioscience) and incubated for 5 minutes at room temperature to block non-specific antigens on the cell surface. Cells were then stained with fluorescence-conjugated antibodies for 30 min at 4 °C. All antibodies are commercially available: CD45-FITC (eBioscience, Cat# 11-0451, 1:200); CD45-APC (eBioscience, Cat# 17-0451-82, 1:200); CD31-FITC (eBioscience, Cat# 11-0311-82, 1:40); CD31-APC (eBioscience, Cat# 170311-82, 1:40); F4/80 PE-Cy7 (biolegend, Cat# 123114, 1:200); F4/80-APC (eBioscience, Cat# 17-4801-82, 1:200); CD11b PE-Cy7 (eBioscience, Cat# 25-0112-82, 1:200); CD11b-APC (eBioscience, Cat# 17-0112-81, 1:200). After staining, cells were washed with PBS to elute excess antibody and then centrifuged at 700 g for 5 min at 4 °C to obtain cells; cell pellets were resuspended in 1 ml of PBS with 1 μl of DAPI and 1 μl of DNase I. DAPI was used to differentiate between dead and live cells, and DNase I was used to avoiding cell adhesion. Flow cytometry analysis was performed using a Beckman CytoFLEX LX, and cell sorting was performed using a SonyMA900, with individual cells collected in a collection buffer (2% FBS in DMEM). 1.RNAseq: The extraction of total RNA from isolated cells was conducted using the RNeasy Micro kit (QIAGEN, GER) following the manufacturer's instructions. Briefly, cells underwent treatment with Buffer RL and 70% ethanol. The resulting mixture was then transferred to a RNeasy MinElute spin column and centrifuged at ≥ 8,000 g for 15 seconds. Subsequent steps involved the addition of Buffer RW1, DNase I, Buffer RPE, and 80% ethanol, followed by additional centrifugation. The RNA-containing RNeasy MinElute spin column was placed in a new 2 ml collection tube, centrifuged with the lid open at full speed for 5 minutes to dry the membrane, and then transferred to a new 1.5 ml tube with RNase-free water. Finally, tubes were centrifuged for 1 minute at full speed to elute the RNA. Qualification and quantification of total RNA were performed using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). High-quality RNA was employed for mRNA library construction. For mRNA library construction, limited RNA (more than 200 pg, high-quality) underwent amplification with oligo-dT and dNTPs. After incubation at 72 ℃ and immediate cooling on ice, reverse transcription to cDNA based on polyA tail was performed. The template was switched to the 5' end of the RNA, and full-length cDNA was generated by PCR. The average molecule length of the PCR product was determined using the Agilent 2100 bioanalyzer instrument. Purified cDNA was fragmented into small pieces with fragment buffer by PCR, and the product was purified and selected by the Agencourt AMPure XPMedium kit (Thermo Fisher Scientific, USA). cDNA quantification was conducted using the Agilent Technologies 2100 bioanalyzer. The double-stranded PCR product, post-QC, was heat denatured and circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) constituted the final library. To ensure the high quality of the sequencing data, two methods were employed for library quantification: determination of the average molecule length using the Agilent 2100 bioanalyzer instrument and quantification of the library using real-time quantitative PCR (qRT-PCR). The final library was amplified with phi29 (Thermo Fisher Scientific, MA, USA) to produce DNA nanoballs (DNB), each containing more than 300 copies of one molecular. DNBs were loaded into the patterned nanoarray, and single-end 50 bases reads were generated on the MGISEQ-2000RS platform (BGI-Shenzhen, China). 2. scRNAseq: Hepatic non-parenchymal cells, isolated from indicated p16-tdT, SnEC-gTracer, or SnMø-cTracer mice, were individually loaded into the 10x Chromium controller to generate GEMs. Subsequently, these GEMs were processed into single-cell 5ʹ gene expression libraries using the Chromium Single Cell 5ʹ Library Kit (v2 Chemistry). All samples were pooled for sequencing on the Illumina NovaSeq 6000 PE150 platform.