Single-cell ATAC sequencing of SIINFEKL-reactive T cells in GL261 murine brain tumors in MHC class II deficient tumor-mircoenvironments
Single-cell ATAC sequencing of SIINFEKL-reactive T cells in GL261 murine brain tumors in MHC class II deficient tumor-mircoenvironments
Mice were anesthetized and perfused with PBS. Brains of tumor-bearing mice were extracted and the tumor bearing hemisphere was cut into pieces and digested with 50 µg/ml liberase (Sigma) at 37°C for 30 minutes and subsequently mashed through a 100 µm and 70 µm strainer (Miltenyi). Myelin was removed using a 30% continuous Percoll (GE Healthcare) gradient and centrifugation at 2700 rpm. Immune cells were isolated as described above. Murine cells were blocked with rat anti-mouse CD16/32 (0.5 μg per well, eBioscience). Dextramer staining was performed according to manufacturer's protocol (Immudex): after blocking, cells were incubated with 1:10 dextramer (PE and APC) dilution in 50 µl PBS for 10 minutes. Respective antibodies in PBS were added in 50 µl total volume and stained for 30 minutes. Following antibodies were used: CD45-BV510, CD3-FITC, MHCII-AF700, CD11b-PEDazzle, CD8-PercpCy5.5. eFluor 780 fixable viability dye (eBioscience) was used according to manufacturer's protocol to exclude dead cells. For scRNA-Seq, cells were pre-incubated for 10 minutes with titrated amounts of TotalSeq C hashing antibodies. Cells were sorted on a BDAria II and BD Aria Fusion through an 85 µM nozzle and 4-way purity. Cells were sorted in 20 µl 0.04% BSA in PBS and kept on ice until processing.  Nuclei isolation was performed using the 10x Nuclei Isolation for Single Cell ATAC Sequencing protocol for low cell input. Cells were resuspended in chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute (Roche), 0.01% Digitonin (Sigma), 1% BSA (Miltenyi) in nuclease free water) and incubated on ice for 3 minutes. Cells were washed, resuspended in Nuclei Buffer (10x). 6.000-10.000 nuclei were used for subsequent single cell ATAC library preparation according to the 10x genomics ChromiumNext GEM Single Cell ATAC v1.1 user guide.