GSM8995005: CD45- cells, Lung, Ctrl; Mus musculus; RNA-Seq
Lung lobes were finely minced and enzymatically digested in PBS containing 5 µg/mL Dispase (Corning, 354235), 0.025 mg/mL DNase I (Roche, 10104159001), and 480 µg/mL Collagenase Type I (Thermo Fisher, 17100017) at 37°C for 30 minutes with intermittent agitation. Single-cell suspensions were obtained using a gentleMACS Dissociator (Miltenyi Biotec). The digested samples were washed with FACS buffer (PBS supplemented with 1 mM EDTA, 25 mM HEPES, and 0.5% BSA), passed through a 70-µm cell strainer, and incubated with 1× RBC Lysis Buffer (BioLegend, 420301) on ice for 5 minutes to remove red blood cells. Following lysis, the cells were blocked with anti-mouse CD16/32 (FcR) antibody (BioLegend, 101301) at 4°C for 1 hour. The FcR-blocked suspensions were then labeled with a biotin-conjugated anti-CD45 antibody at 4°C for 1 hour, followed by incubation with anti-biotin microbeads (Miltenyi Biotec, 130-090-485) for 30 minutes. CD45⁻ cells were subsequently isolated using LS columns (Miltenyi Biotec) and washed with FACS buffer prior to downstream processing. Library was prepared using Chromium Next GEM Single Cell 5' Reagent Kit v2 (10x Genomics) following manufacturer's instruction.