title: GSM8490509: ND539 Recurrent Tumor Model SOX4KO; Homo sapiens; RNA-Seq
library_construction_protocol: Followed manufacturers protocol (10x single cell RNA-seq v3.1) scRNA-seq libraries were generated using a Chromium Single-Cell 3' v3.1 Library and Gel Bead Kit (10x Genomics) following the manufacturer's protocol. Briefly, for recurrent mouse tumor and chronic antigen stimulation assays, CD8+ T cells were sorted by FACS and washed with ice-cold PBS + 0.04% BSA. After washing, 15,000 cells were loaded onto the chromium controller to generate single-cell gel beads in emulsion. For patient samples, approximately 35,000-100,000 CD3+ T cells were sorted by FACS and cells were washed twice with ice-cold PBS + 0.04% BSA and spun down at 600g for 5 minutes. After washing, cells were resuspended in up to 45 ml of ice-cold PBS + 0.04% BSA. Five microliters of cell suspension were mixed with 5 ml of trypan blue, and cell numbers were counted via hemacytometer under the microscope. Approximately 10,000-16,000 cells were loaded to create the gel bead in emulsion. Following reverse transcription, gel beads in emulsion were disrupted and barcoded complementary DNA was isolated and amplified. After fragmentation, end repair, and poly A tailing, sample indexes were added and amplified following the manufacturer's protocol. The final libraries were quality control checked and sequenced on an Illumina NextSeq 500.  Patient 04 was sequenced with a 75-cycle kit with parameters Read 1: 28, Read 2: 56, Index 1: 8, Index 2: 0 and patient 05 with a 150-cycle kit with parameters Read 1: 28, Read 2: 91, Index 1: 8, Index 2: 0. After fragmentation, end repair, and poly A tailing, sample indexes were added and amplified following the manufacturer's protocol. The final libraries were quality control checked and sequenced on an Illumina NextSeq 500.  Patient 04 was sequenced with a 75-cycle kit with parameters Read 1: 28, Read 2: 56, Index 1: 8, Index 2: 0 and patient 05 with a 150-cycle kit with parameters Read 1: 28, Read 2: 91, Index 1: 8, Index 2: 0.