GSM6123290: splenic slices transplant; Mus musculus; RNA-Seq
Mice were perfused through portal vein cannulation by normal saline solution. Liver tissues were removed and minced with ophthalmic scissors in DMEM medium, and enzymatically digested using the liver dissociation kit (Miltenyi Biotech , 130-105-807). Cell suspensions were filtered through a 100 μm nylon mesh, centrifuged at 50 g for 3 min  at 4 ºC, and the supernatants were further centrifuged at 320 g for 8 min to obtain non-parenchymal liver cells. Isolation of hepatic immune cells were performed through a 30% Percoll (GE Healthcare, 17-0891-01) . Primary CD45+ non-parenchymal liver cells  were further sorted by staining with antibodies antibody  for mouse CD45 (Biolegend, 103112). Cells were sorted using FACSAria (Becton, Dickinson). Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion (GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5'end (that is, the 3'end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer's protocol (Chromium Single Cell 3ʹ v3). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina) using 2x150 chemistry.