GSM8005168: TME_Tom negative; Mus musculus; RNA-Seq
The mice were anesthetized with avertin (tribromoethanol) and further systemically perfused with PBS to remove the circulating cells in tumors. Tumors were collected, minced, and incubated for 20 minutes in 50 mg/mL Liberase DH (Roche), 10 mM HEPES, 2 kunitz/mL DNase in DMEM at 37oC and passed through 18-gauge needles followed by 21-gauge needles with a 20 min incubation in between. Cells were filtered through a 70 mm cell strainer and centrifuged at 400 g for 3 min. The pellet was resuspended into 10 mL ACK lysis buffer for 3 min at room temperature and washed with wash buffer. Cells were treated with an FcR blocking reagent on ice for 10 min, stained with fluorescence-labeled antibodies at 4oC for 30 min, washed, and analyzed or sorted using FACS SORPAria (BD Biosciences). For the scRNA-seq library construction, we followed the manufacturer's instructions of 10x Genomics Chromium Single Cell 3′ Reagent Kit v3.1.