Illumina NovaSeq 6000 paired end sequencing; IL-2wt and IL-2nα differentially reshape the TME
Five days after the last dose (3mg/kg) of IgG, IL-2wt and IL-2nα mutein, MC38 tumor (four mice from each group) were dissected from mice and digested as above mentioned. Tumor-infiltrating leukocytes (TILs) were isolated by Percoll gradient and CD45+ leukocytes were enriched by CD45 (TIL) MicroBeads (Miltenyi, 130-110-618). Samples from the same group were labeled with different TotalSeq™ anti-mouse Hashtag (Biolegend) for 10x Genomics platform. Three samples from each group with higher cell viability were selected. 10,000 cells from each sample were mixed into a single sample and resuspended with a concentration of 1,000 cells/ul. Single-cell RNAseq library was constructed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1.  Single cell suspensions were first partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs). Full-length cDNA tagged with a 16nt 10x barcode and barcoded DNA from the cell surface protein Feature Barcode were generated within each individual GEM during RT reaction. Purified DNA after RT reaction was amplified for 11 cycles and size selection was used to separate the amplified cDNA molecules for 3ʹ Gene Expression and Cell Surface Protein library construction.