GSM8074571: Pt3_Aph-VDJ; Homo sapiens; RNA-Seq
1. For isolated TIL: Tumor was minced and carefully mixed in digestion medium with collagenase (type II and type IV), hyaluronidase and DNAase (Thermo Fisher Scientific). Then undigested tumor was filtered from the cell suspension to generate a single-cell suspension. Intact cells were enriched with a Ficoll-Hypaque (GE Healthcare Bio-Sciences) gradient and viable tumor cells were enumerated by trypan blue exclusion. The tumor digested was stained with anti-human CD3 and Zombie NIR. Isolated TIL was sorted as the CD3+ Zombie NIR- population and sent for single cell sequencing.  2. For cultured TIL: Samples were stained with anti-human CD3 and Zombie NIR, and flow sorting was conducted to sort CD3+Zombie NIR- TIL for single cell sequencing. The cell suspension was loaded onto the 10X Genomics Chromium Single Cell Controller (10X Genomics, Inc., Pleasanton, CA) at a concentration of about one thousand cells per microliter and the single cells, reagents, and 10x Genomics gel beads were encapsulated into individual nanoliter-sized Gelbeads in Emulsion (GEMs).  Revers transcription of poly-adenylated mRNA was performed inside each droplet, and cDNA generated from cells encapsulated with a 10X Genomics Chromium NextGEM Single Cell V(D)J Reagent Kit was enriched using VDJ primers provided in the kit.  A portion of the DNA library generated from antibody-labeled cells was amplified using primers provided by 10X Genomics to prepare a cell surface protein library, and the cDNA, feature barcoding and VDJ-enriched libraries were completed in bulk reactions using the 10X Genomics Chromium NextGEM Single Cell 3' or 5' V(D)J Reagent Kits.