design: Single cell RNA-seq of Panc02-H7-Fluc model with vehicle tobemstomig, nivolumab and relatlimab-like treatment
library_construction_protocol: Mice were sacrificed, and tumor tissue was isolated. The tumor tissue was disrupted and then digested in an enzyme mix containing RPMI with 10 mg/ml DNAse and 0.25 mg/ml Liberase for 30 minutes at 37°C. The resulting tissue mix was filtered to obtain a single-cell suspension. Lymphocytes were mechanically isolated from draining lymph nodes, filtered, and resuspended to a single-cell suspension. The resulting single-cell suspensions from tumors and lymph nodes were stored in liquid nitrogen in pZerve freezing medium. After thawing a batch of samples, the cells were stained with a mix of FACS and oligo-labelled antibodies and sorted for CD3+T cells before performing scRNA-seq. Prior to sorting, the cells were washed with PBS, and cell number and viability were evaluated. Approximately 1 million cells per sample were resuspended in PBS and incubated with a Fc blocking reagent. The mix of FACS and oligo-labelled antibodies was added, and after incubation and washing, the cells were resuspended for sorting. The cell number and viability of the sorted cells were determined, and a total of 10,000 viable cells per sample were loaded into the 10x Genomics Chromium Connect Instrument. cDNA and library preparation was performed according to the manufacturer's indications using the 10x Genomics Chromium Next GEM Automated Single Cell 5' Reagent Kits v2 with TCR and feature barcoding.