GSM4987067: LLC_TCR_profiling_tet-_rep2; Mus musculus; RNA-Seq
tumors cell suspensions were generated using a Tumor Dissociation Kit (Miltenyi) and the gentleMACS Dissociator (Miltenyi) using the m-TDK-2 program. The resulting cell suspension then passed through a 70 micron filter before lymphocytes were isolated using Lympholyte M cell separation media (Cedarlane), per manufacturer's instructions. Cells were then enriched for CD45+ cells using CD45 microbeads, mouse (Miltenyi) and the MultiMACS Cell24 Separator Plus (Miltenyi). Cells were then stained in PBS using Live/Dead Fixable Near-IR Dead Cell Stain Kit (1:100, ThermoFischer), H-2Kb MuLV p15E Tetramer (1:10, MBL International), and TrueStain FcX Plus (anti-mouse CD16/32) Antibody (1:1000, BioLegend). Samples were then stained for CD45 (1:100, Biolegend) and CD8 (1:10, MBL International) in MACS Buffer. Samples were pooled and analyzed using a MA900 FACS instrument (Sony), using single-colour compensation controls and fluorescence-minus-one controls for setting gates. Live, cD45+, CD8+, and p15E+ cells were sorted into a RMPI with 50% FBS solution. p15E- cells were also sorted. RNA was  isolated using an RNeasy Micro kit (Qiagen). TCR profiling from RNA was performed using the SMARTer Mouse TCR a/b Profiling Kit (Takara) llumina sequencing primers were added in a semi-nested approach during TCR amplification; libraries were purified, pooled and then sequenced on an illumina Miseq platform