Gentlemacs dissociator and tumor infiltrating lymphocyte kit

For single-cell preparation, tumor tissue was dissociated with the gentleMACS Dissociator and Tumor infiltrating Lymphocyte Kit (Miltenyi Biotec) according to the manufacturer's protocol. The cells were then cryopreserved in liquid nitrogen until use. All samples showed a viability of around 90% on average after thawing. We performed 5′ gene expression profiling on the same single-cell suspension using the Chromium Single Cell V(D)J Solution from 10x Genomics according to the manufacturer's instructions. Up to 8,000 cells were loaded onto a 10x Genomics cartridge for each sample. Cell-barcoded 5′ gene expression Libraries were constructed and sequenced at a depth of approximately 50,000 reads per cell using the NovaSeq 6000 platform (Illumina).

For single-cell preparation, tumor tissue was dissociated with the gentleMACS Dissociator and Tumor Infiltrating Lymphocyte Kit (Miltenyi Biotec) according to the manufacturer's protocol. The cells were then cryopreserved in liquid nitrogen until use. All samples showed a viability of around 90% on average after thawing. scRNA-seq libraries were generated using the Chromium Single Cell 3 Library & Gel Bead Kit v3 (10 × Genomics) following the manufacturer's instructions. Briefly, the Chromium instrument was used to separate single cells into gel bead emulsions that facilitated the addition of cell-specific barcodes to all cDNAs generated during oligo-dT–primed reverse transcription. As a result, a cell barcoding sequence and Unique Molecular Identifier were added to each cDNA molecule. Libraries were constructed and sequenced at a depth of approximately 50,000 reads per cell using the HiSeq2500 platform (Illumina).