GSM8994902: Lung CD140b+ cells, FOXF2, Wild Type, rep1; Mus musculus; OTHER
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. Each reaction was performed with approximately 30,000 cells. To begin, cells were incubated at room temperature for 15 minutes with magnetic beads coated with concanavalin A (Vazyme, N515). Following this, primary antibodies specific to either FOXF2 or H3K4me2 were added, and the mixture was rotated overnight at 4°C. The next day, secondary antibodies were introduced and allowed to incubate for 30 minutes at room temperature. Excess antibodies were removed using a Triton-based wash buffer, after which the pG-Tn5 adapter complex (diluted 1:25) was added, and the samples were incubated for 1 hour at room temperature. For CUT&Tag processing, cells were resuspended in 50 µL of tagmentation buffer consisting of 1 mL Triton-300 wash buffer and 10 µL of 1 M MgCl₂, and incubated at 37°C for 1 hour. In the case of H3K4me2 CUTAC, cells were instead resuspended in 50 µL of a DMF-based tagmentation buffer (10 mM TAPS, 5 mM MgCl₂, 20% DMF, and 0.05% Triton X-100) and incubated at 37°C for 20 minutes. After tagmentation, DNA was extracted using SDS–Proteinase K treatment, and libraries were generated via PCR amplification with the TruePrep CUT&Tag Amplification Mix (Vazyme, TD612), employing Illumina i5 and i7 indexing primers. The amplified products were purified with VAHTS DNA Clean Beads (Vazyme, N411) and sequenced using the Illumina NovaSeq 6000 platform.