GSM6998229: WT_LN_1_GEX; Mus musculus; RNA-Seq
For the isolation of TILs, tumor tissue was dissociated with collagenase D (2.5mg/ml) at 37 degrees Celsius for 20 min, followed by centrifugation through a discontinuous Percoll gradient (GE Healthcare). 1,000 living CD8+ T cells (CD45+ TCRß+ CD8) were FACS-sorted immediately into TCL buffer (QIAGEN) supplemented with 1% ß-mercaptoethanol (Sigma Aldrich). To obtain cells from draining lymph nodes, lymph nodes were mashed through a 40 µm strainer. In the EAE experiment, mice (8-12 weeks old) sex-matched were immunized subcutaneously into the flanks with an emulsion containing the MOG35-55 peptide (100µg/mouse, Genemed Synthesis) and M. tuberculosis H37Ra extract (5mg/ml, Becton Dickinson) in CFA (200ul/mouse, Becton Dickinson). Pertussis toxin (100ng/mouse, List Biological Laboratories) was administered intravenously on days 0 and 2. For the isolation of lymphocytes from EAE mice, mice were sacrificed, and perfusion was performed intracardially with PBS. To obtain cells from  the CNS, the forebrain and cerebellum were dissected and together with the spinal cord were flushed out with PBS using hydrostatic pressure. CNS tissue was cut into small pieces with a razor blade and digested with collagenase D (2.5mg/ml, Roche Diagnostics) at 37 degrees Celsius for 20 min. Mononuclear cells were isolated by passing the tissue through a 40 µm strainer, followed by centrifugation through a Percoll gradient (37% and 70%). Lastly, the mononuclear cells were obtained from the interphase, washed, and used for further analysis. The cell populations of interest were sorted by FACS for sequencing. For cell hashing, the cells were incubated for 30 min on ice in flow buffer with the TotalSeqTM-antibody pool (Biolegend) at 1:1000 final dilution for each antibody, followed by a total of three washes with flow buffer. Following the manufacturer's instructions (10x Genomics), the samples were then separated into droplet emulsions using the Chromium Single Cell 5' V2 Solution. The library preparations of the scRNA-seq, including TCR-seq and 5' feature barcoding libraries were performed according to manufacturer's instructions (10x Genomics) using the NextGem 5' v2 Dual Index protocol. Libraries were pooled to 4nM.