GSM9756172: Lung cells, LPS + Reg3g; Mus musculus; RNA-Seq
Mouse lungs were harvested, minced on ice, and digested with Liberase (Sigma-Aldrich) at 37 °C for 15 min. Following enzymatic dissociation, cells were pelleted by centrifugation at 500 × g for 5 min, and residual erythrocytes were removed using RBC Lysis Buffer (Beyotime). Single-cell suspensions were filtered through a 40-μm cell strainer and stained for 30 min at 4 °C in the dark using fluorophore-conjugated antibodies against CD45 (PE-Cy5), Ly6G (AF700), F4/80 (PE), CD11b (PerCP-Cy5.5), and CD11c (BV605) (BioLegend). DAPI was added 10 min before sorting to exclude nonviable cells. Fluorescence-activated cell sorting was performed on a BD FACSAria Fusion flow cytometer, and lung macrophages were identified as DAPI⁻CD45⁺Ly6G⁻F4/80⁺CD11b/CD11c⁺ cells. Sorted cells were collected in culture medium supplemented with 20% FBS. To ensure sufficient cell yield for downstream scRNA-seq analysis, each biological replicate consisted of pooled cells from three mice. Sorted cells were washed and resuspended in 1× PBS containing 0.04% BSA. Cell concentration was adjusted to 700–1,200 cells/μL and viability was confirmed by acridine orange/propidium iodide staining and CountStar counting. Cells were loaded into the Chromium Controller (10x Genomics, PN-120270) on a Chromium GEM-X Single Cell 3′ Chip (10x Genomics, PN-1000690) to generate single-cell gel beads in emulsion (GEMs) according to the manufacturer's protocol (10x Genomics, CG000732). cDNA amplification and library construction were performed using the Chromium GEM-X Single Cell 3′ Reagent Kit v4 (10x Genomics, PN-1000691). Constructed libraries were quality-controlled using the Agilent dsDNA 915 Reagent Kit (Agilent, DNF-915-K0500) and quantified using the VAHTS Library Quantification Kit for Illumina (VAHTS, NQ101). Libraries were sequenced on the Illumina NovaSeq™ X Plus platform with 2 × 150 paired-end kits. Read 1 (28 bp) was used for cell barcode and UMI, and Read 2 (91 bp) for transcript sequence, with a minimum sequencing depth of 20,000 reads per cell.