title: GSM7790638: CD8+ NRT cells, scTCR-seq analysis, tcr repertoire; Homo sapiens; OTHER
library_construction_protocol: A pool of ~750,000 barcodes are sampled separately to index each cell's transcriptome. It is done by partitioning cell harvested after neoantigen-specific stimulation into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNAs share a common 10x Barcode. GEMs are generated by combining barcoded Single Cell 5' Gel Beads, a Master Mix with cells, and Partitioning Oil on Chromium Next GEM Chip G. To achieve single cell resolution, cells are delivered at a limiting dilution, such that the majority (~90 – 99%) of generated GEMs contains no cell, while the remainder largely contain a single cell. Immediately following GEM generation, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing an Illumina R1 sequence (read 1 sequencing primer), a 16 nt 10x Barcode, a 10 nt unique molecular identifier (UMI), and  13 nt template switch oligo (TSO) are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents and poly(dT) RT primers. Incubation of the GEMs produces 10x Barcoded, full-length cDNA from polyadenylated mRNA. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size prior to 5' Gene Expression library construction. primers, a sample index, and Illumina R2 sequence (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and Sample Index PCR. The final libraries contain the primers priming sites used in Illumina sequencers 10X5'+VDJ and single-cell RNA-Seq