GSM5525976: T8.D32.Fat2 [ATAC-seq]; Mus musculus; ATAC-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, 5e3 P14 cells were sorted from the spleen and blood and 5e3 IV- P14 cells were sorted from the IEL, kidney, salivary gland, fat, and liver into PBS + 5% BSA. To further improve sample purity, 1e3 cells from each sample were resorted into 1x TCL lysis buffer + 1% 2-mercaptoethanol. For each replicate, cells from 10-15 mice were pooled and then 2e4 cells were sorted into PBS + 5% BSA and spun down at 500 x g for 20 min at 4°C. The cell pellet was then resuspended in 25 µL lysis buffer and then spun down at 600 x g for 30 min at 4°C. The nuclei pellet was resuspended in 25 µL transposition reaction mixture containing Tn5 transposase from Nextera DNA Sample Perp Kit (Illumina) and incubated at 37°C for 30 min. The transposase-associated DNA was then purified using the Zymo DNA clean-up kit. To amplify the library, the DNA was first amplified for 5 cycles using indexing primers from the Nextera kit and NEBNext High-Fidelity 2X PCR master mix. To reduce the PCR amplification bias, after the first 5 cycles, 5 uL of amplified DNA was used to do qPCR to determine the number of cycles for the second round of PCR. The total amplified DNA was then size selected to fragments less than 800 bp using gel purification. The size of the pooled library was examined by tapestation.