Disaggregation of Tumor Tissue Samples

Tumour tissue samples were disaggregated into single-cell suspension as previously described for synovial tissue [4 (link)]. In brief small fragments (~1–2 mm³) of tumour tissue were generated by dissecting with forceps or surgical scissors. The cut fragments were cryopreserved for subsequent disaggregation; they were transferred to a cryovial (1.5 ml; Nalgene) containing 1 ml of CryoStor® CS10 for viable freezing. Tumour fragments were thawed, washed and dissociated by enzymatic digestion using RPMI media with Liberase™ TL enzyme preparations (100 μg/ml; Roche) and DNase I (100 μg/ml; Roche) prior to single-cell analysis. Single-cell suspensions were assessed for cell quantity and cell viability. To remove non-viable cells before library construction, 7-AAD viability dye (#A1310, ThermoFisher) was used.