title: GSM3457010: ccRCC_2_ft_gex_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Tumor tissues were obtained through the Stanford Tissue Bank from patients undergoing surgical resection at Stanford University Medical Center (SUMC). All experiments utilizing human material were approved by SUMC's Institutional Review Board and performed under protocol #28908. Written informed consent for research was obtained from donors prior to tissue acquisition. Samples were confirmed to be tumor by pathological assessment at SUMC. Cellular suspensions were loaded on a ChromiumTM Single Cell Controller instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. Single-cell RNA-Seq libraries were prepared using the ChromiumTM Single Cell 5' Library & Gel Bead Kit (P/N 1000006, 10x Genomics). GEM-RT was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; P/N 1851197):  53?°C for 45 min, 85?°C for 5?min; held at 4?°C and stored at -20?°C. The GEMs were shipped to 10x Genomics on dry ice, then broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; P/N 37002D) . Barcoded, full length cDNA was amplified using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98?°C for 45 s; cycled 13 ×: 98?°C for 20?s, 67?°C for 30?s, and 72?°C for 1?min; 72?°C for 1?min; held at 4?°C. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6 × SPRI; Beckman Coulter; P/N B23318) .  Barcoded, full-length V(D)J segments were enriched from amplified cDNA with primers specific to TCR constant regions. The target enrichment 1 was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98?°C for 45 s; cycled 10 ×: 98?°C for 20?s, 67?°C for 30?s, and 72?°C for 1?min; 72?°C for 1?min; held at 4?°C. The target enrichment 1 was cleaned up with the SPRIselect Reagent Kit (0.8 × SPRI).  The target enrichment 2 was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98?°C for 45 s; cycled 10 ×: 98?°C for 20?s, 67?°C for 30?s, and 72?°C for 1?min; 72?°C for 1?min; held at 4?°C. The target enrichment 2 was cleaned up twice with the SPRIselect Reagent Kit (0.5 × and 0.8 × SPRI). The enrichment primers can be found in Supplementary Table 4. 5' gene expression and enriched libraries were constructed using the reagents in the ChromiumTM Single Cell 3'/5' Library Construction kit (P/N 1000020). For 5' gene expression library construction, these steps were followed: (1) fragmentation, end repair and A-tailing; (2) post fragmentation, end repair and A-tailing cleanup with SPRIselect; (3) adaptor ligation; (4) post ligation cleanup with SPRIselect; (5) sample index PCR and cleanup. For the enriched library construction, these steps were followed: (1) fragmentation, end repair and A-tailing; (2) adaptor ligation; (3) post ligation cleanup with SPRIselect; (4) sample index PCR and cleanup. The barcode sequencing libraries were quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms P/N KK4824). Sequencing libraries were loaded at concentrations on sequencers with the read configuration as specified in Table S5.