GSM9288671: p312awt_IMM_GEX; Mus musculus; OTHER
Lung immune and epithelial cells were isolated using a modified enzymatic digestion protocol from a previous study with minimal modification (17). Mice were euthanized and lungs were perfused and lavaged with digestion buffer composed of RPMI-1640 supplemented with elastase (Worthington), dispase II (Sigma-Aldrich), and DNase I (Sigma-Aldrich). Lung tissues were then excised, minced into small fragments, and further digested in RPMI-1640 containing liberase (Sigma-Aldrich) and DNase I at 37°C for 30 minutes. Following enzymatic digestion, single-cell suspensions were obtained by passing the mixture through a 100 μm cell strainer and red blood cells were lysed using ACK lysis buffer. To enrich for specific cell subsets, cells were stained with surface markers targeting CD11b⁺, EpCAM⁺, and CD11c⁺ populations using a customized innate immune panel. CD3⁺ T cells were enriched following FACS protocol. Cells were sorted using a FACSAria cell sorter (BD Biosciences) after staining with oligo antibodies (Table S2). CD11b⁺, EpCAM⁺, CD11c⁺, CD3⁺ cells and the non-sorted fraction at a 1:1:1:1:6 ratio and resuspended in cold PBS supplemented with 1% BSA (Miltenyi Biotec) and 0.5 U/μL RNase inhibitor (Sigma-Aldrich) for scRNA-seq. CD11b⁺, EpCAM⁺, CD11c⁺ and the non-sorted fraction at a 1:1:1:7 ratio and resuspended in BD OMICS Guard™ buffer (BD Biosciences, Cat. 570911).Spatial transcriptomics Lung tissues from C57BL/6 mice were freshly harvested, embedded in optimal cutting temperature (OCT) compound, and immediately snap-frozen in liquid nitrogen. Samples were stored at −80 °C until sectioning. scRNA-seq analysis To simultaneously assess whole-transcriptome and targeted surface protein expression at the single-cell level, BD Rhapsody™ single-cell multi-omics technology was employed. Mouse lung cells were thawed and resuspended in FACS buffer prior to dextramer-based flow cytometric sorting for antigen-specific T cells. Sorted cells were collected into staining buffer at a concentration of 200–800 cells/μL in ~600 μL per sample. Approximately half of the collected cells were loaded onto the BD Rhapsody Cartridge (BD Biosciences, Cat. #633733) using the associated BD Rhapsody Cartridge Reagent Kit (Cat. #633731). Cells were captured in microwell arrays with barcoded beads, followed by cell lysis, bead retrieval, cDNA synthesis, template switching, and Klenow extension. Full-length cDNA containing unique molecular identifiers (UMIs) and cell barcodes was PCR-amplified. Whole-transcriptome was prepared using the BD Rhapsody Whole Transcriptome Amplification Kit (Cat. #633801) following the manufacturer's instructions. scATAC-seq analysis Cryopreserved cells stored in BD OMICS-Guard™ buffer (Cat. #570911) for less than 48 hours were centrifuged at 800 × g for 5 minutes to remove the preservation buffer. Without additional washing, cells were resuspended in BD sample buffer at ~800 cells/μL for downstream ATAC-seq processing. Splint beads were prepared using a custom splint oligonucleotide specific to V3 capture beads provided by BD Biosciences. Nuclei were isolated following a modified protocol (62). Briefly, cell pellets were lysed and resuspended in Nuclei Buffer using a wide-bore pipette, aiming for a concentration of 10,000 nuclei/μL. In situ tagmentation was performed by incubating the nuclei with a Tagmentase mix that targets accessible chromatin regions, generating DNA fragments with preloaded adapter sequences. An aliquot of tagmented nuclei was stained with DyeCycle™ Green (1 mM) and counted using the BD Rhapsody™ Scanner. Single-nucleus capture, cell lysis, and genomic DNA processing were performed according to the BD Rhapsody manufacturer's protocol. DNA fragments were captured via splint-oligo-bonded TSO (template-switch oligo) strands. Ligase was used to ligate the bead-bound oligos to the tagmented DNA, followed by gap filling and extension using a specific enzyme mix. Final denaturation removed DNA templates from beads, and Illumina-compatible adapters and sample indices were introduced during amplification. Libraries were prepared with the following sequencing parameters: Read 1: 50 cycles, Read 2: 50 cycles, Index 1: 8 cycles, and Index 2: 60 cycles. Sequencing data were analyzed using the BD Rhapsody ATAC Large Input pipeline (v2.2.1) on VELSERA cloud platform. Processed output files were used for secondary analysis and visualization using the Signac package (v1.14.0). In brief, fragment files were imported and used to create a Seurat object with a chromatin assay via the CreateChromatinAssay function. Spatial transcriptomics Lung tissues from C57BL/6 mice were freshly harvested, embedded in optimal cutting temperature (OCT) compound, and immediately snap-frozen in liquid nitrogen. Samples were stored at −80 °C until sectioning. Spatially resolved single-nucleus transcriptomic profiling was performed using the Curio Trekker Single-Cell Spatial Mapping Kit (Cat. Nos. SK017 and SK020; Takara Bio). Briefly, 25 μm cryosections of mouse lung tissue were placed onto Trekker 10 × 10 mm glass tiles, each embedded with a monolayer of uniquely DNA-barcoded microparticles (beads). Upon exposure to ultraviolet (UV) light, the spatial DNA barcodes were photocleaved and internalized into the overlying tissue. Following UV exposure and barcode incorporation, the tissue was dissociated from the tile into single-nucleus suspensions. These spatially tagged nuclei were then loaded onto the BD Rhapsody™ Single-Cell system (BD Biosciences) for single-nucleus capture and transcriptomic profiling.