title: GSM6670638: murine liver, control; Mus musculus; RNA-Seq
library_construction_protocol: Mice were anesthetized using pentobarbital sodium and the livers were perfused with a two-step perfusion method through portal vein. Pre-warmed first perfusion solution (ionized water containing 8 mg/mL NaCl, 0.402 mg/mL KCl, 0.094 mg/mL NaH2PO4·2H2O, 0.2865 mg/mL Na2HPO4·12H2O, 2.383 mg/mL HEPES, 0.19 mg/mL EGTA, 0.3528 mg/mL NaHCO3 and 0.901 mg/mL glucose, pH = 7.4) was perfused at a rate of 6 mL/min for about 8 minutes, followed by the perfusion of pre-warmed second perfusion solution (DMEM medium containing 0.6 mg/mL type VI collagenase, 0.1 mg/mL trypsin inhibitor, 1 mg/mL DNase I, 5 mg/mL BSA, 5 mM CaCl2, pH = 7.4) at a rate of 5 mL/min for 6 minutes. The perfused liver was subsequently dissected using forceps and dissociated with the digestion solution (DMEM medium containing 5 mg/mL BSA and 5 Mm EDTA) for 10 minutes to obtain single-cell suspension. Hepatocytes and non-parenchymal cells (NPCs) were then separated through a centrifugation at 50 g for 3 minutes. For each group, equal amount of the same type of cells from distinct mouse were mixed. Dead cells in hepatocytes and non-parenchymal cells were removed separately using Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101) for higher sequencing quality. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.