GSM9079139: I36H2_spleen_LCMVarm_D7_allT_P14_SMARTA_mouse0127_FBC; Mus musculus; OTHER
Single-cell suspensions of spleens and lymph nodes were prepared by mechanical disruption, followed by ACK-lysing buffer. For tissue preparations, siIEL were prepared by excision of Peyer's patches and removal of luminal contents. Then the entire tissue section was cut longitudinally, then cut laterally into 1 cm pieces, which were then incubated at 37°C for 30 minutes in Hanks' balanced salt solution with 2.4 mg ml−1 of Hepes, 2.1 mg ml−1 of sodium bicarbonate, 8% bovine growth serum and 0.154 mg ml−1 of DTE (EMD Millipore). After collecting the siIEL sample, RPMI with 1.2 mg ml−1 of Hepes, 292 µg ml−1 of L-glutamine, 1 mM MgCl2, 1 mM CaCl2, 5% FBS and 100 U ml−1 of collagenase (Worthington) was added to the SI pieces at 37°C for 30 minutes for siLPL isolation. The SG and lung were cut into small pieces then incubated with shaking at 37°C for 30 minutes (SG) or 1 hour (lung) in the same RPMI-collagenase solution as listed previously for the siLPL. Prostate tissue was dissected from the bladder and seminal vesicles, cut into small pieces, the incubated shaking at 37°C for 30 minutes in the same RPMI-collagenase solution as listed previously. Bone marrow was isolated by harvesting the femurs and tibias and pushing out the bone marrow with a syringe. Lymphocytes from non-lymphoid tissues were further enriched using a 44%/67% Percoll density gradient. Cells were incubated with FC block and stained with Live/Dead, CD3, CD45.2 and CD45.1 on ice prior to sorting. Libraries were prepared according to the 10x Genomics 5'v2 protocol for GEX, VDJ, and FBC