GSM9151063: E0771_Ctrl_TIL; Mus musculus; RNA-Seq
Single-cell suspensions from E0771 tumors with indicated treatment were prepared by using mouse tumor dissociation kit (Cat#130-096-730, Miltenyi Biotec) in combination with the gentleMACS Octo Dissociator. In brief, fresh E0771 tumors were resected and minced with sterile scissors into approximately 1- to 2- mm3 pieces, then transferred into gentleMACS C Tube with 5 mL enzyme mix prepared as recommended in the protocol (5 mL enzyme mix for maximal 1g tumor tissue). Program '37C_m_TDK_2' was used with the heating function. After termination of the program, samples were resuspended after adding 10 mL of DMEM/F12 medium supplemented with 2% FBS and passed through a 40 mm cell strainer (BD Biosciences). Cell suspension was centrifuged at 350g for 5 minutes and resuspended in PBS. Cells were then stained with zombie violet fixable viability dye (Cat#423114, BioLegend) on ice for 15 minutes followed by a 20-minute incubation with TruStain FcX. Subsequently, cells were incubated with APC anti-mouse CD45 antibody (Cat#103112, BioLegend)on ice for 30 minutes. After washing wash twice with cell staining buffer (Cat# 420201, BioLegend), FACS was conducted on BD FACSAria Fusion Cell Sorter (BD Biosciences) to isolate live CD45+ cells. Sorted CD45+ cells were suspended at a concentration of 5,000 live cells/L with cell suspension buffer provided in the PIPseq T20 3ʹ Single Cell RNA Kit V4. A total of 40,000 cells puls 40 units of RNase inhibitor were added into pre-templated instant partitions (PIPs). Samples were processed following the workflow of capture and lysis, mRNA isolation, complementary DNA (cDNA) synthesis, cDNA amplification, library preparation, library pooling, and sequencing according to the user's guide (Doc ID:FB0002130, Fluent BioSciences).