title: Illumina NovaSeq 6000 paired end sequencing; Single-cell RNAseq of HA-specific CD8 T cells isolated from the hypothalamus and the spleen of mice expressing the model antigen HA in orexinergic neurons
library_construction_protocol: Naïve HA-specific CD8+ T cells were purified from  the spleen and lymph nodes of CL4-TCR F1 mice. Purified HA-specific CD8+ T cells were then cultivated with irradiated syngeneic splenocytes in the presence of HA512-520-peptide, IL-2, and IL-12. After five days of culture, living cells were collected. 3×106 activated CD8+ T cells were adoptively transferred via i.v. in female adult (5 – 7 week old) Orex-HA mice. At day 30 post-transfer, the mice were sacrificed and the HA-specific CD8+ T cells (CD11b- NK1.1- CD45.2+ CD8+ TCRb+ CD45.1+) were isolated by FACS sorting (BD FACSAria Fusion) from the spleen and hypothalami of the 13 Orex-HA mice. Immediately after cell sorting, cells were subjected to scRNA-seq processing (Chromium Single Cell 3ʹ Reagent Kits v3, CG000183 Rev C) according to the manufacturer's instructions.  Libraries were constructed using the 10X Genomics, Chromium Single Cell 3' v3 kit  including : - Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075;  Chromium Single Cell B Chip Kit, 48 rxns PN-1000073 and Chromium i7 Multiplex Kit, 96 rxns PN-120262; according to manufacturer's instructions.