GSM8757497: Thymic iNKT cells, Prkd2/3-deficient, scTCR-seq; Mus musculus; OTHER
Thymocytes were resuspended in 2%FBS/RPMI1640 medium and stained with anti-mouse TCRb-FITC and alpha-Galactosylceramide (a-GalCer)-loaded mouse CD1d tetramer-APC and anti-APC microbeads. Thymic iNKT cells were enriched by MACS magnetic separator (Miltenyi). Thereafter cells were passed through 0.45 um mesh, stained with propium iodide (PI) and purified for TCRβ+CD1d tetramer+PI– cells by a cell sorter (SH800S, Sony). Purified thymic iNKT cells were washed and suspended in PBS (–) containing w/v 0.04% BSA and applied to scRNA/TCR-seq analysis. For scATAC-seq analysis, nuclei were isolated from purified thymic iNKT cells according to the manufacturer's instruction (10x Genomics). For scRNA-TCR-seq, single cell library was prepared using Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 and Chromium Single Cell V(D)J Enrichment Kit for mouse T cell (10x Genomics) following the manufacturer's instructions. After reverse transcription, cDNA was amplified for 14 cycles, and up to 50 ng of cDNA was used for construction of gene expression and TCR libraries. For scATAC-seq, single cell library was prepared using Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit (10x Genomics) following the manufacturer's instructions.