GSM9217277: MMTV-PyMT_3; Mus musculus; RNA-Seq
Tumors were collected, mechanically dissociated, and resuspended in 2.35 mL of RPMI 1640 medium supplemented with 100 µL of Enzyme D, 50 µL of Enzyme R, and 12.5 µL of Enzyme A (Miltenyi Biotec #130-096-730). Tissue dissociation was performed using the gentleMACSTM Octo Dissociator with Heaters (Miltenyi Biotec #130-096-427) according to the manufacturer's program (37C_m_TDK_1). Following dissociation, cell suspensions were filtered through a 70 μm mesh and washed with FACS buffer (1% FBS in PBS). Cells were centrifuged at 500 × g for 5 minutes and resuspended in 500 μL of FACS buffer. For flow cytometry analysis, cells were stained with anti-CD45 antibody (BD, #550994) and Fixable Viability Stain 450 (BD, #562247) to assess viability. CD45+ viable cells were sorted using a BD FACSAria™ III cell sorter, washed in PBS, and resuspended at a concentration of 1 × 10⁶ cells/mL. Single-cell suspensions were subsequently loaded onto a Chromium Controller (10x Genomics, Pleasanton, CA) using the Single Cell 3' Library and Gel Bead Kit v3 (10x Genomics) for droplet-based encapsulation and library preparation.