GSM9226069: TILs with 156-WT TCRs; Homo sapiens; RNA-Seq
one million PC3-A2-PAP cells in 100 µL of 1X PBS were injected subcutaneously into the right flank of NSG mice to establish tumor xenografts. Six weeks after tumor engraftment, 5 million TCR-engineered PBMCs were administered intravenously via the tail vein. Five animals were included in each group. Three out of five animals with similar sizes of tumors were sacrificed 10 days post-injection. Tumors were excised, cut into 2-3 mm pieces, and digested with 1 mg/mL Collagenase Type I (Gibco) and 1 mg/mL Dispase (Gibco), supplemented with Rock inhibitor (Tocris Bioscience) and DNase I (Roche, Sigma), at 37°C for one hour. This was followed by a 5-minute treatment with TrypLE (Gibco). The processed cells were then filtered through a 40 µm filter and stained with anti-human CD3 (clone SK7, eBioscience), NGFR (Biolegend), and anti-mouse CD45 (clone 30-F11, eBioscience). Cells identified as DAPI-muCD45-CD3+NGFR+ were single-cell sorted into 1X PBS with 0.04% bovine serum albumin (BSA). Cells from the same group (n = 3) were pooled and subsequently processed by the UCLA Technology Center for Genomics and Bioinformatics (TCGB) core for 10X Genomics GEM-X 3' Gene Expression v4 library preparation and next-generation sequencing on the Illumina Novaseq X platform. Library construction was performed following the protocol of Chromium GEM-X Single Cell 3' (polyA) v4 Gene Expression Guide (https://www.10xgenomics.com/support/universal-three-prime-gene-expression/documentation/steps/library-prep/chromium-gem-x-single-cell-3-v4-gene-expression-user-guide)