title: GSM9530783: Intrahepatic leukocytes, CCl4-4, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Sample preparation. Mouse livers were harvested after 6 weeks of twice weekly carbon tetrachloride (CCl4) or Olive oil administration. In situ tissue digestion was performed by perfusing the liver with collagenase P (COLLP-RO, Millipore Sigma) at a concentration of 64 mg/mL in a balanced salt solution. Digested livers were then homogenized in a petri dish in a solution containing 0.15 mg/mL DNase I (11284932001, MilliporeSigma).  The intrahepatic leukocyte fraction was obtained by 25%-50% Percoll gradient(28). Cells were processed following the 10x Genomics protocol for cell/nuclei fixation (CG000782). Briefly, suspensions containing up to ten million cells were spun down at 350 rcf for 5 minutes at 4ºC, then resuspended in 1 mL of Fixation Buffer (4% Formaldehyde, 1X Fix and Perm Buffer – 10x Genomics PN-2001301). Cells were fixed for 24 hours at 4ºC. To halt fixation, cells were spun down at 850 rcf for 5 minutes at room temperature and quenched with 0.5 mL of Quenching Buffer (1X Quench Buffer – 10x Genomics PN-2001300). Cell concentration and viability were assessed via AO/PI staining using a Cellometer K2 Cell Counter (Nexcelom), then processed according to the 10x Genomics Flex protocol for hybridization. Sample hybridization. All hybridizations followed 10x Genomics recommendations (CG000787 Rev B). Hybridizations were set up in 40 µL of hybridization mix with 10 µL of Mouse WTA probes (10x Genomics PN-2001275-2001278). Hybridizations were performed at 42ºC for 16-24 hours. Afterward, samples were diluted in Post-Hyb Wash Buffer and measured via AO/PI staining with a Cellometer K2 Cell Counter. Pooled hybridized cells were washed three times in Post-Hyb Wash Buffer for 10 minutes at 42ºC. After washing, cells were resuspended in Post-Hyb Resuspension Buffer, filtered through a Miltenyi Biotec 30 μm filter, and measured to determine the volume required for the GEM-X Flex Gene Expression run. Before probe hybridization, an equal number of cells from each fixation were pooled (pre-hybridization normalization) to ensure equal contribution per sample. Gene Expression Flex Run. For Gel Beads-in-emulsion (GEM) generation, we followed the 10x Genomics protocol and guidelines on cell and reagent volumes based on targeted cell recovery. We aimed for 20,000 cells per sample. For the probe mixing strategy experiment, we targeted 80,000 cells per pool with equal contribution from each sample, except for the differential pooling strategy, where contributions varied. After loading the GEM-X FX Chip on the Chromium X, GEMs were recovered and processed as per 10x Genomics instructions. The product was then pre-amplified and indexed for library construction. Libraries were quality-checked using the fragment analyzer.Sequencing. Libraries were sequenced on the Illumina Nova-Seq X 1.5B platform according to 10x Genomics recommended settings, with a minimum of 10,000 read pairs per cell. For experiments involving probe mixing strategies, sequencing was conducted at a depth of 10,000 read pairs per sample, yielding 80,000 cells per library.