title: GSM7114877: CD276-8HTM-BBz CAR_3_CSP; Homo sapiens; OTHER
library_construction_protocol: 3E+06 of luciferase labeling JR cells (an aggressive RMS cells) were resuspended in Matrigel (Corning) after washing and intramuscularly injected into the NSG mice. 13 days later, 2.5E+06 FGFR4 or CD276 CAR T cells were intravenously injected into above tumor bearing NSG mice. Tumor infiltrating lymphocytes (TILs) from the RMS JR model were purified from tumors 11 days after T cell administration using 40% and 70% percoll (Cytiva, Cat# 17089102) gradient separation and then sorted viable human CD45+ lymphocytes on BD FACSAria Fusion and BD FACSAria UV. After washing with CITE-seq antibody staining buffer (DPBS with 0.05% BSA), sorted TILs were counted, resuspend at a concentration of 1E+06/50μL and stained with TotalSeq-C human “hashtag” antibodies (hashtag 5 or hashtag 6) for each mouse from 5 CAR T cells treated groups, allowing identification of different replicates from each group in the analysis. Next, above TILs were individually stained with a cocktail of a TotalSeq-C human lyophilized panel (BioLegend) of 17 surface proteins. After 3 washes, TILs were resuspended in PBS and counted and then replicates in each group were combined before proceeding immediately to single-cell immune profiling using a Chromium Single Cell 5' Solution v2 platform system (10× Genomics). 10X Genomics 5' single-cell gene expression and cell-surface protein tag libraries were prepared as instructed by the 10X Genomics user's guides. Both libraries were sequenced on an Illumina NextSeq 500 individually.