GSM8818972: SingleCell Vista KO VDJ TCRseq; Mus musculus; OTHER
B16 melanoma tumors were pooled and digested to obtain single cell suspensions. Tumor infiltrating CD8  and CD4 T cells were stained with anti-CD8 antibody and purified by FACS sorting. 10e4 sorted viable T cells  were loaded into a Chromium Controller (10X Genomics). Viable sorted T cells were washed three times before being processed using 10X genomics Feature barcoding protocol and workflow with v3.1 3' technology. The single-cell RNAseq libraries are prepared by the Genomics Core facility at Lerner Research Institute. The single-cell RNAseq libraries are prepared according to the manufacturer's Chromium Single Cell 3ʹ v3.1 with Feature Barcode technology for Cell Surface Protein (CSP) protocol. Briefly, cDNA for the 3' gene expression library was initially synthesized from poly-adenylated mRNA, and DNA for the CSP library was synthesized from the cell surface protein feature Barcode. A minimum of 1 nanogram of cDNA was used to begin fragmentation, followed by end-repair, adapter ligation, and sample indexing. Each sequencing library including the CSP library was tagged with a dual index (10-nucleotide long each) sample barcode that identifies from which sample a sequence was derived. In addition, a 12-base unique molecular indexes (UMIs) were introduced to reduce duplicate reads. Constructed libraries are pooled and quantified using a Quantabio Q cycler. NovaSeq 6000 RapidRun flow cells are used to sequence a minimum of 20,000 read pairs per cell for the Gene expression library and a minimum of 5,000 read pairs per cell for the Cell surface protein library using specifications recommended by the manufacturer (10X Genomics, document # CG000317). Separate feature barcoding libraries sequenced targeting 5,000 reads per cell at 10,000 cells per sample and were mapped to cells using cellranger software. Antibody counts were compiled with gene expression data in Seurat V4.0 software and normalized via CLR normalization methods for plotting.