GSM9032526: NT; Mus musculus; RNA-Seq
For cell sorting, the lungs from three mice per condition were harvested, pooleand enzymatically digested with 0.1 mg/mL DNase I (Sigma-Aldrich), 1.5 U/mL Dispase II (Gibco), and 200 U/mL Collagenase Type IV (Gibco) in RPMI+ GlutaMAX (Gibco) for 30 min at 37°C under agitation. Samples were filtered using 70 µm cell strainer and washed with RPMI+ GlutaMAX (Gibco) with 10% FBS. After digestion, red blood cells were lysed with ACK lysis buffer (Gibco) for 5 min at room temperature and washed with PBS.27,000 monocytes and tumor associated macrophages (defined as CD45+ CD11b+ ECFP+ EGFP+ CD4- CD8- Ly6G- SiglecF-), 3,000 alveolar macrophages (defined as CD45+ SiglecF+ CD11b- ECFP+ CD4- CD8- Ly6G-), 10,000 CD8+ T cells (defined as CD45+ CD8+ CD4- CD11b- Ly6G- SiglecF-), and 20,000 CD4+ T cells (defined as CD45+ CD4+ CD8- CD11b- Ly6G- SiglecF-) were sorted in PBS 0.5% BSA from a pool of three tumor-bearing mice per condition. After sorting, all cell preparations were resuspended at 106cells/mL in PBS containing 0.5% BSA and loaded onto a Chromium Controller (10X Genomics). Samples were processed for single-cell encapsulation and cDNA library generation according to the manufacturer's instructions using the 10x Genomics Single Cell 3′v2 Reagent Kit (for mouse). The library was  sequenced using a NovaSeq 6000 (Illumina).