GSM8927136: mouse perfusion isolated hepatocyte,P7-L,snRNA-seq; Mus musculus; RNA-Seq
Mouse hepatocytes isolation was performed as previously reported. Mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol and then perfused through the inferior vena cava with 10 mL of pre-warmed (37°C) phosphate-buffered saline (PBS) containing 1 mM EGTA. The portal vein was cut at the start of perfusion to allow blood clearance. The liver was then perfused and digested with 10 mL of pre-warmed 1 mg/mL collagenase IV (Sigma-Aldrich, C5138) in RPMI 1640 medium (Gibco, 21870076). The digested liver tissue was collected into PBS and filtered through a 70 μm cell strainer. The cell suspension was centrifuged at 50 × g for 2 minutes at 4°C, and the cell pellets were resuspended in RPMI 1640 medium supplemented with 1% fetal bovine serum (FBS). To enrich for viable hepatocytes, Percoll (GE, 17-0891-09) diluted in PBS was added to the cell suspension, followed by centrifugation at 50 × g for 2 minutes. The cell pellets were then collected, washed once, and resuspended in RPMI 1640 medium supplemented with 1% FBS. Smart-seq3xpress scRNA-seq experiments was performed according to the published procedure(Hagemann-Jensen et al., 2022). Briefly, purified single hepatocytes were manually picked with a microcapillary pipette under a microscope and transferred into the 384-well PCR plates containing 0.3 μL of lysis buffer and 3 μL of silicone oil (Merck, 317667). The mRNA was reverse transcribed into cDNA using 0.2 μL of dispensed RT mix. The cDNA was then amplified using 0.6 μL PCR mix. The library of each cell was constructed separately using the TruePrep DNA Library Prep Kit (Vazyme, TD501) Smart-seq3xpress scRNA-seq experiments was performed according to the published procedure(Hagemann-Jensen et al., 2022). Briefly, purified single hepatocytes were manually picked with a microcapillary pipette under a microscope and transferred into the 384-well PCR plates containing 0.3 μL of lysis buffer and 3 μL of silicone oil (Merck, 317667). The mRNA was reverse transcribed into cDNA using 0.2 μL of dispensed RT mix. The cDNA was then amplified using 0.6 μL PCR mix. The library of each cell was constructed separately using the TruePrep DNA Library Prep Kit (Vazyme, TD501)