GSM8415434: sheep hepatocyte,G50_70,scRNA-seq; Ovis aries; RNA-Seq
Slaughtered sheep livers were cut into small cubes and stored in Belzer UW Cold Storage Solution at 4°C and transported to the laboratory. For isolation of sheep hepatocytes, livers were dissected into 1 mm3 cubes and digested with RPMI 1640 medium containing 2.5 mg/mL collagenase IV (Sigma‒Aldrich, C5138) and 2.5 mg/mL collagenase II at 37°C for 20 minutes. The cell suspension was centrifuged at 50 × g for 3 minutes at 4 °C, and then the cell pellets were resuspended and washed twice with RPMI 1640 medium containing 10% FBS at 4°C. 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)