GSM8967922: Prostate_D34_Cd3e_RNA; Mus musculus; RNA-Seq
Prostate tissue was dissected from the bladder and seminal vesicles, cut into small pieces, the incubated shaking at 37°C for 30 minutes in 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). Lymphocytes from the prostate were separated on a 44%/67% Percoll density gradient. CD3e+ T cells from the prostates and spleens of mice were profiled by single-cell RNA-seq 34 days after initial LCMV infection. 5x104 CD3e+ T cells from 7 pooled mouse prostates were sorted into RPMI + Glutamax supplemented with 10% bovine growth serum, 1% HEPES, 1% penicillin and streptomycin, and 0.1% 2-Mercaptoethanol. Samples were spun down at 500 RCF for 5 minutes and resuspended in 0.04% bovine serum albumin. Samples were then loaded into a Chromium Chip B (10X Genomics) and partitioned into gel bead-in-emulsions in a chromium controller (10X genomics). scRNA-seq libraries were generated using 10X Genomics 5'v2 with feature barcoding for cell surface protein and immune receptor mapping (CG000330) and sequenced on a HiSeq 4000.