GSM8266678: TOXOTRM2 Latency 52dpi CD103-neg; Mus musculus; RNA-Seq
Whole brains (including the pia mater, but excluding the dura mater and arachnoid tissue which remain attached to the skull) were collected in complete RPMI supplemented with 10% vol/vol FCS, homogenized using a glass Potter and digested for 45 min at room temperature in HBSS with collagenase D (1 mg/mL) and deoxyribonuclease (DNase) I (20 ug/mL). After digestion, cells were suspended in 30% Percoll (GE Healthcare) and centrifuged at 1590g for 30 min without brake.  Myelin and debris were removed, brain leukocytes were recovered from the interface. Before scRNA-seq, CD103-negative and CD103-positive subpopulations of Kb-OVA dextramer+ CD8+ T cells were separated by Facs-sorting.  For CITE-seq, brain cell suspensions were incubated for 45 min at RT with the following barcoded dextramers diluted in complete RPMI: Kb-SIINFEKL (Immudex cat # JD2163-PfBC, barcode fBC0074: CGGTCTTAGTCGCGC, dilution 1:50), Ld-HPGSVNEFDF (Immudex cat # JG5820-PfBC0301, barcode fBC0301: CGGCCTCGCGACGAC, dilution 1:50), control Kb-SIYRYYGL (Immudex cat # JD2164-PfBC, barcode fBC0068: TTGCGCGGCGTCGTA, dilution 1:50), and then surface labelled for 30 min at 4°C with TotalSeq-C0182 anti-mouse CD3 (clone 17A2, dilution 1:50, Biolegend, cat # 100263) and TotalSeq-C0002 anti-mouse CD8 (clone 53-6.7, dilution 1:50, Biolegend, cat # 100785) diluted in 'Facs buffer' (PBS, 2mM EDTA, 0.5% FCS). For scRNA-seq, single-cell libraries were generated after Facs-sorting using the Chromium Controller Instrument and Chromium Single Cell 3' Library & Gel Bead Kit v3 (10X Genomics). For CITE-seq, single-cell libraries were generated with Chromium Next GEM Single Cell 5' Kit v2 and 5' Feature Barcode Kit (10X Genomics).