GSM8968135: Rep 1: RT Day 3, FTY720 Day 3, RT+FTY720 Day 3 CITE-seq HTO; Mus musculus; OTHER
Tumors were manually dissociated using surgical scissors and the contents were transferred into Miltenyi Biotec gentleMACS C tubes containing enzyme digestion mixture, consisting of with 250 U/ml collagenase IV, 30 U/ml DNase I, 5 mM CaCl2, 5% heat-inactivated FBS, and HBSS. Dissociated tumors in collagenase cocktail were shaken at 37°C for 30 minutes. Samples were washed with RPMI containing 10% FBS and 2 mM EDTA. Digested contents were filtered through 70 µm cell strainers and washed in RPMI containing 10% FBS and 2 mM EDTA. Samples were enriched magnetically using CD4/CD8 TIL MicroBeads. Samples were then stained with viability dye, CD90.2-Alexa Fluor 700, and Totalseq hashtag antibodies. Kaede green or red live CD90.2+ cells were sorted using a Symphony S6 cell sorter with a 100µM nozzle. Library construction was performed using the 10X Genomics Chromium GEM-X Single Cell 5' Reagent v3 with Feature Barcode technology for Cell Surface Protein protocol (CG000734).