title: scRNA- and TCR-Seq of mouse T cells from WT and TIGIT KO mice at steady state and upon LCMV infection
design: scRNA- and TCR-Seq of mouse T cells from WT and TIGIT KO mice at steady state and upon LCMV infection
library_name: KO_spl_LCMV 6_TCR_p
library_construction_protocol: Spleen and lung single cell suspensions were obtained from naïve and LCMV Clone 13 infected (10^6 ffu, day) 10 wild-type (C57BL6) and TIGIT KO (B6.129(Cg)-Foxp3tm4(YFP/icre)Ayr/J x Tigitfl/fl) mice. CD3+ T cells were MACS purified using the CD3 cell isolation kit (negative selection, BioLegend) and then FACS sorted using a BD FACSAria III 5L (BD Bioscience).  Single-cell suspensions of spleen were generated by mechanical disruption in RPMI 1640 (Gibco) supplemented with 10% FCS (Corning or Capri), penicillin and streptomycin (100 U/ml, Gibco) and 1% L-glutamine (Gibco). Red blood cells were lysed using ACK lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH: 7,4) for 3-5 minutes. Single-cell suspensions of the lung were generated by enzymatic tissue digestion at 37 °C with 0.2 mg/ml Deoxyribonuclease I (Sigma) and 2.4 mg/ml Collagenase type I (Gibco) and mechanical disruption using the GentleMACS. Lung immune cells were separated from epithelial and parenchymal cells, which were excluded with 30% Percoll solution gradient (GE Healthcare). mice were either left naive or infected with 10`6 ffc LCMV clone 13 and organs were harvested on day 10 of infection Sorted CD3+CD4+Foxp3+ and CD3+Foxp3- cells were mixed at a ratio of approximately 1:3 and labelled for each mouse with anti-mouse Hashtag reagents (TotalSeq™-C301, 302, 303, 304, 305 anti-mouse Hashtag 1, 2, 3, 4, 5 antibodies, respectively, BioLegend) before pooling .  Cells were loaded onto a Chromium Next GEM chip G (10X Genomics) and were lysed for reverse transcription and complementary DNA (cDNA) amplification in the Chromium Controller (10X Genomics) following the 10x 5' (v2) Dual Index protocol (https://cdn.10xgenomics.com/image/upload/v1722286086/support-documents/CG000330_Chromium_Next_GEM_Single_Cell_5_v2_Cell_Surface_Protein_UserGuide_RevG.pdf) Library construction was performed following the 10x 5' (v2) Dual Index protocol: Cells were lysed for reverse transcription and complementary DNA (cDNA) amplification in the Chromium Controller (10X Genomics). The poly-adenylated mRNAs were then reverse-transcribed inside each Gel Bead. Full-length cDNA along with cell barcode identifiers were PCR-amplified and sequencing libraries were prepared . The following kits were used:  Chromium Next GEM Single Cell 5' Kit v2 Library Construction Kit Chromium 5' Feature Barcode Kit Chromium Single Cell Mouse TCR Amplification Kit Chromium Next GEM Chip K Single Cell Kit