GSM8994972: Lung CD140b+ cells, KO, rep2; Mus musculus; RNA-Seq
Lung lobes were finely minced and digested with an enzyme solution 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) in PBS at 37°C for 30 minutes with periodic agitation. Single-cell suspensions were generated using a gentleMACS Dissociator (Miltenyi Biotec). The digested tissues were washed with FACS buffer (PBS with 1 mM EDTA, 25 mM HEPES, and 0.5% BSA), filtered through a 70-µm cell strainer, and treated with 1× RBC Lysis Buffer (BioLegend, 420301) for 5 minutes on ice to eliminate red blood cells. The samples were then blocked with anti-mouse CD16/32 (FcR) antibody (BioLegend, 101301) at 4°C for 1 hour. The FcR-blocked single-cell suspensions were incubated with biotin-conjugated anti-CD140b (PDGFRβ) antibody at 4°C for 1 hour, followed by anti-biotin microbeads (Miltenyi Biotec, 130-090-485) for 30 minutes. PDGFRβ+ cells bound to microbeads were enriched using LS columns (Miltenyi Biotec) and washed with FACS buffer. RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs)