title: Illumina NovaSeq 6000 paired end sequencing; Single-cell sequencing of tissue-resident CD4+ T cells that drive compartmentalized immunopathology in the CNS
design: Single-cell sequencing of tissue-resident CD4+ T cells that drive compartmentalized immunopathology in the CNS
library_construction_protocol: CD4 T cells were isolated via FACS seven days post addition of tamoxifen. We employed intracranial (i.c.) infection of adult GFAP:GP mice with rLCMV-GP33 as an experimental model of transient viral infection of the brain to generate GP33-specific tissue-resident memory CD8+ T cells (TRM). Cross-reactivity of TRM with a CNS-restricted self-antigen was modeled by the TAM-inducible expression of LCMV glycoprotein (GP) antigens in a CNS-restricted cell population (astrocytes). For depletion of circulating CD8+ T cells or CD4+ T cells, mice were treated intraperitoneally twice over a 3 days interval with 200 μg of rat-anti-mouse-CD8α-depleting antibody (BioXCell, BE0117), 250 μg of rat-anti-mouse-CD4 –depleting antibody (home-made, clone YTS191) or isotype control (BioXCell, BE0090).Anti-CD8 was administered 7 and 4 days before start of TAM while anti-CD4 was administered 4 and 1 day prior start of TAM. For relapse experiments, mice were treated intraperitoneally with 200 μg of mouse-anti-mouse-CD8α -depleting antibody (Ab00166-2.0, Absolute Antibody) or isotype control (Ab00166-2.3, Absolute Antibody) 7 and 4 days before each TAM administration. According to 10x 5' protocol According to 10x 5' protocol