GSM8279430: CD45+ cells, GEX; Homo sapiens; RNA-Seq
Tumor samples were dissociated into single cells by a semi-automated combined mechanical/enzymatic process. The tumor tissue was cut into pieces of (2-3mm) in size and transferred to C Tubes (Miltenyi Biotech) containing a mix of Enzymes H, R and A (Tumor Dissociation Kit, human; Miltenyi Biotech). Mechanical dissociation was accomplished by performing a program (37C_h_TDK_1) on the gentleMAC Octo Dissociator with Heaters (Miltenyi Biotech). Single cell suspensions generated from ccRCC tumor samples were FACS sorted on a SONY MA900 sorter (SONY) for viable human TILs (Zombie- mCD45- hCD45+, BioLegend). Sorted cells were stained with a distinct barcoded antibody (Cell-Hashing antibody, TotalSeq-C, Biolegend). After washing, the stained cells were resuspended in 0.4% BSA in PBS at a concentration of 2,000 cells per μL, then loaded onto a single lane (Chromium chip K, 10X Genomics) followed by encapsulation in a lipid droplet (Single Cell 5′kit V2, 10X Genomics) followed by cDNA and library generation according to the manufacturer's protocol. 5′ mRNA library was sequenced to an average of 50,000 reads per cell, V(D)J library and protein (hashtags) library sequenced to an average of 15,000 reads per cell, all using Illumina Novaseq.