GSM7050303: scRNAseq_IgG_day07_rep1; Mus musculus; OTHER
CD4+ T cells were prepared from spleen using mouse CD4+ T cell Isolation Kit (Miltenyi Biotech) and AutoMACS cell separator (Miltenyi Biotech) according to manufacturer's instructions. Enriched CD4+ T cells, which included PbT-II cells, were incubated with a panel of TotalSeqTM -C antibodies (BioLegend) and surface antibody markers in PBS + 1% BSA: TotalSeq™-C0238 Rat IgG2a for isotype control (clone RTK2758), TotalSeq™-C0078 anti-CD49d (clone R1-2), TotalSeq™-C0198 anti-CD127 (clone A7R34), and TotalSeq™-C0250 anti-KLRG1, as well as anti-CD4 (clone GK1.5), anti- TCR β chain (clone H57-597), anti-TCR anti-CD45.1 (clone A20), and 7AAD. Stained CD4+ T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5' v2 protocol). PbT-II cells (CD4+CD45.1+TCR+) were sorted using FACSAria II cell sorter (BD Biosciences). FACS-sorted cells were collected in PBS + 1%BSA after washing, and resulted in >99% purity and >98% viability in all samples. Single-cell RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index; 10X Genomics) according to the manufacturer's instructions. Libraries were quantified using Bioanalyzer High Sensitivity Chip (Agilent). Gene expression libraries and cell surface protein libraries were pooled for library construction and sequencing was performed using MGISeq-2000RS.