GSM8505162: CD8, rep2; Mus musculus; Hi-C
Mice: 6-week-old female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Experiments using C57BL/6J mice were performed in accordance with a protocol (number 3063), which was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Princeton University. Isolation and preparation of CD4 and CD8 T cells from mouse spleen: Mice were euthanized by intraperitoneal injection of ketamine/xylazine at a supratherapeutic dose. Spleens were collected and placed into 5 mL of serum-free Dulbecco's Modified Eagle Media (DMEM, Thermo Fisher Scientific, Waltham, MA, 11995081). Individual spleens were placed on 100-μm strainers then mechanically dissociated using the plunger of a 3 mL syringe. The strainer was washed with 5 mL of serum-free DMEM in which the splenocytes were subsequently resuspended. The single cell suspension was centrifuged at 1,300 rpm for 5 min at 4°C, then the pellet was resuspended in 1x BD Pharm Lyse lysis buffer (BD Biosciences, Franklin Lakes, NJ, 55599) and incubated at room temperature in the dark for 15 min. Lysis was quenched using DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. The splenocyte suspensions were spun at 1,300 rpm for 5 min at 4°C, then washed with FACS buffer (1% (v/v) FBS in PBS). The cells were counted using 0.4% trypan blue solution (Thermo Fisher Scientific, 15250061) and a hemocytometer, then resuspended at a concentration of 1 x 107 cells/mL. An aliquot of cells was set aside as an unstained control. Another aliquot of cells was set aside to generate the live/dead control in which half of the cells are heat shocked at 95°C for 5 min, set on ice for 2 min, and combined with the live cells.  Antibody staining and fluorescence-activated cell sorting: The following fluorophore-conjugated antibodies were used for flow cytometry: CD45-FITC (clone 30-F11, 1.25 μg/mL, BioLegend, San Diego, CA 103108), CD3-Alexa700 (clone 17A2, 1 μg/mL, Invitrogen, Waltham, MA, 56-0032-82), CD4-PE-Cy7 (clone RM4-5, 1 μg/mL, Invitrogen, 25-0042-82), CD8-BV510 (clone 53-6.7, 1 μg/mL, BioLegend, 100752). The antibody cocktail was prepared in FACS buffer, then incubated with cells for 30 min at 4°C in the dark. The splenocyte suspensions were washed 2x in FACS buffer. To serve as a viability marker, DAPI (1 μg /mL, Sigma-Aldrich, St. Louis, MO, D9542) was directly added to antibody-stained cells and the live/dead control 15 min prior to FACS acquisition. Compensation controls were prepared using AbC Total Antibody Compensation Bead Kit (Invitrogen, A10497). The FACSAria Fusion Flow Cytometer (BD Biosciences) or the MA-900 cell sorter (Sony Biotechnology, San Jose, CA) was used to perform cell sorting and acquired data were analyzed using BD FACSDiva or MA900 software. For collection of each T cell subset, the samples were gated as follows: for CD3+ T cells, CD45+ CD3+; for CD4+ T cells, CD45+ CD3+ CD4+ CD8- ; and for CD8+ T cells, CD45+ CD3+ CD4- CD8+. Cells were sorted into FACS buffer and maintained at 4°C.