scRNA-seq analysis of CD4 T cells reactive toward alpha-zein, helicobacter hepaticus, or unknown small or large intestinal antigens
scRNA-seq analysis of CD4 T cells reactive toward alpha-zein, helicobacter hepaticus, or unknown small or large intestinal antigens
Cells from each animal were resuspended in 100 μl staining buffer (PBS with 4% FBS and 2mM EDTA) along with 0.2 micrograms of a unique hashing antibody to mark each category: 155861 TotalSeq C0301 anti-mouse Hashtag 1, 155863 TotalSeq C0302 anti-mouse Hashtag 1, 155865 TotalSeq™-C0303 anti-mouse Hashtag 3, 155867 TotalSeq™- C0304 anti-mouse Hashtag 4, 155869 TotalSeq™-C0305 anti-mouse Hashtag 5, 155871 TotalSeq™-C0306 anti-mouse Hashtag 6. Along with hashing, cells were stained with surface antibodies at 1:400 dilution, and appropriate tetramers at 1:100 dilution, followed by 3 washes 6 with 200 μl staining buffer. Pooled cells were subsequently sorted on a FACSAriaII (BD Biosciences) using a 70-micron nozzle, operating FACSDiva software, with post-sort analysis performed on FlowJo 10.8.1 (Tree Star) After passing through a 40-micron filter and resuspending in PBS, approximately 43,000 final cells were loaded onto three lanes of 10x Genomics Chip K, following manufacturer protocols. RNA, HTO, and TCR libraries were constructed strictly following the Chromium Next GEM Single Cell 5' dual index protocol (10X Genomics kit 1000265). After passing through a 40-micron filter and resuspending in PBS, approximately 43,000 final cells were loaded onto three lanes of 10x Genomics Chip K, following manufacturer protocols. RNA, HTO, and TCR libraries were constructed strictly following the Chromium Next GEM Single Cell 5' dual index protocol (10X Genomics kit 1000265).