GSM9155138: HSC. DCV, rep1; Homo sapiens; RNA-Seq
Cocultures were gently washed with Versene and incubated in Versene at 37°C for 20–25 minutes to loosen cell contacts. After washing with pre-warmed DMEM/F12, cells were incubated with a pre-warmed collagenase mix (2.0 mg/mL collagenase, 1.0 mg/mL dispase, 100 U/mL DNase I, and 0.2% DMSO in HBM) at 37°C for 35–40 minutes, with gentle pipetting to aid dissociation. The cell suspension was then collected in Versene and centrifuged at 400 × g for 5 minutes. Cells were resuspended in Versene and incubated again at 37°C for 45 minutes, resulting in mostly single cells. To separate hepatocytes from HSCs, the single-cell suspension was blocked with 1% BSA on ice for 1 hour, stained with mouse anti-ASGR1 antibody, and incubated with anti-mouse IgG magnetic microbeads. Magnetic separation was performed at 4°C. Hepatocytes were collected from the magnet-bound fraction, while HSCs were collected from the flow-through after a second round of hepatocyte depletion. Library preparation was performed according to the manufacturer's instructions (Single Cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded, along with partitioning oil and gel beads, into the chip to generate gel bead-in-emulsions (GEMs). The poly-A RNA from lysed cells within each GEM was reverse transcribed into cDNA, incorporating an Illumina Read 1 primer sequence, a Unique Molecular Identifier (UMI), and a 10x barcode. The pooled, barcoded cDNA was then purified using Silane DynaBeads, amplified by PCR, and size-selected using SPRIselect reagent to enrich for fragments ranging from 300 to 700 base pairs for subsequent library construction. The returned Fastq files of DMSO and DCV samples (DMSO-1, DMSO-2, DCV-1, and DCV-2) were analyzed with Cell Ranger and converted to Seurat objects by running the corresponding scRNA-seq codes that have been published in github and provided by the Cleveland Clinic Center for Immunotherapy and Precision Immuno-Oncology (CITI) on the Cleveland Clinic's internal High Performance Computing (HPC) platform. The final integrated Seurat object generated from the four Fastq files was uploaded to BioTuring for analysis.