title: GSM7469176: 2C_Flank2; Mus musculus; RNA-Seq
library_construction_protocol: Tumor-bearing mice were injected retro-orbitally with fluorescently-labeled anti-CD45 antibodies (CD45-IV) 3 minutes prior to euthanasia to differentiate tumor- and lung-infiltrating immune cells from circulating immune cells. Spleens and lymph nodes were dissected from mice and physically dissociated through a 70 µm filter to generate single cell suspensions. Splenocyte suspensions were lysed with ACK lysis buffer (Gibco) for 3 minutes to deplete red blood cells. Subcutaneous tumors and tumor-bearing lungs were dissected from mice, weighed, and collected in 5 mL DMEM (Gibco) containing the human tumor dissociation kit (Miltenyi) enzymes. Tumors were dissociated using the gentleMACS Octo Dissociator (Miltenyi). Following the digestion, tumor or lung pieces were mashed through a 70 µm filter with a 1 mL syringe plunger to generate a single cell suspension. The dissociated cells were washed 2 times with PBS and were layered over Ficoll (GE). Cells were spun over Ficoll at 450 g for 30 minutes with the lowest settings of acceleration and brakes. The layer at the interface of Ficoll and PBS, which contained the majority of the live immune cells, was collected and washed with PBS. Cell suspensions were washed twice with cold PBS + 0.5% BSA + 2mM EDTA and then enriched for tumor-resident immune cells by performing negative selection with anti-PE microbeads (Miltenyi Biotec) followed by positive or negative selection with anti-CD45 microbeads (Miltenyi Biotec). 2C T cells were adoptively transferred to tumor-bearing mice as described above. Seventy-two hours after transfer, TdLN cells were isolated and prepared for FACS. 2C T cells were FACS-sorted directly into TRIzol reagent and RNA-extracted. RNA library preparation and sequencing were performed at the KI Genomics Core / MIT BioMicro Center. Sequencing was performed using the HiSeq 2000 sequencing system (Illumina).