GSM5526039: T8.D32.Liv3 [scRNA-seq]; Mus musculus; RNA-Seq
To identify CD8+ T cells in the vasculature of non-lymphoid tissues (small intestine, kidney, salivary gland, fat, and liver), 3 µg of CD8α (53-6.7) conjugated to APC eFluor780 was injected by i.v. into mice three minutes prior to sacrifice, as has been previously described. Cells labeled with low to no CD8α antibody were considered to be outside of the vasculature. Single-cell suspensions of splenocytes were prepared by mechanical disaggregation followed by treatment with ACK lysis buffer. Blood samples were treated with ACK lysis buffer. Small intestine intraepithelial lymphocytes were prepared through the removal of Peyer's patches and the luminal contents. The small intestine was then cut longitudinally and into 1 cm pieces, then incubated at 37°C for 30 min in HBSS with 2.1 mg/mL sodium bicarbonate, 2.4 mg/mL HEPES, 8% bovine growth serum, and 0.154 mg/mL of dithioetheritol (EMD Millipore). The kidneys, salivary glands, fat, and liver were minced into small pieces and then incubated in RPMI with 1.2 mg/mL HEPES, 292 µ/mL L-glutamine, 1 mM MgCl2, 1 mM CaCl2, 5% fetal bovine serum, and 100 U/mL collagenase (Worthington) at 37°C for 30 min. Lymphocytes from the small intestine, kidney, salivary gland, and liver were separated on a 44%/67% Percoll density gradient. For each replicate, cells from 10-15 mice were pooled and then sorted for bulk RNA-seq. 32 days after initial infection with LCMV, 1e4 P14 cells were sorted from the spleen and blood and 1e4 IV- P14 cells were sorted from the IEL, kidney, salivary gland, fat, and liver into T cell media. Cells were then spun down and media removed prior to starting library preparation. Libraries were prepared using the Chromium Single Cell 3' kit with v3 chemistry according to manufacturer's instructions.