GSM8626613: LSEC WT; Mus musculus; RNA-Seq
Mice were deeply anesthetized with Isolfluran (Baxter) and sacrificed by cervical dislocation. Livers were perfused in situ via the portal vein with a 0.05% collagenase/amino acid/saccharide solution (C2674, Sigma-Aldrich). Livers were dissected and mechanically disrupted. Liver tissue from three mice was pooled and digested at 38 °C in collagenase/Gey's balanced salt solution (G9779, Sigma-Aldrich). Then, the tissue was filtered through a 250 μm mesh. Non-parenchymal cells were separated by a 19.3 % Nycodenz (1002424, Axis-Shield, Alere Technologies) gradient. Subsequently, magnetic-activated cell sorting (MACS) was used with anti-CD146 MicroBeads (ME-9F1, 130-092-007, Miltenyi Biotech) to purify endothelial cells. LSEC suspensions were adjusted to a concentration of approximately 1,000 cells/µL in PBS to ensure optimal capture efficiency for 10x Genomics Chromium Next GEM technology. Single-cell suspensions were loaded into the 10x Genomics Chromium Controller (10x Genomics) to generate Gel Bead-in-Emulsions (GEMs), following the manufacturer's instructions (10x Genomics protocol number: CG000317 Rev A). A targeted cell recovery of 10,000 cells was used to ensure sufficient capture while minimizing doublet formation. Inside each GEM, reverse transcription was performed using reagents from the 10x Genomics Chromium Single Cell 3' Reagent Kit v3.1. Barcoded mRNA transcripts were converted into cDNA within the emulsion droplets. Following reverse transcription, cDNA was purified with Silane Dynabeads (Thermo Fisher Scientific), and the cDNA libraries were amplified by polymerase chain reaction (PCR) to generate sufficient material for sequencing. Amplified cDNA was subjected to fragmentation, end repair, and A-tailing using the Chromium Single Cell 3' Library Construction Kit. Adapter ligation and final amplification were performed to generate sequencing-ready libraries. Sample quality was assessed using an Agilent Bioanalyzer (Agilent Technologies) to ensure library integrity and appropriate fragment sizes.