Tumor preparation for RNA-seq

RNA-seq, flow cytometry, and single-cell RNA-seq were performed on tumors from separate experiments. For RNA-seq, whole tumors and lymph nodes were surgically resected, and the surrounding tissue was removed and immediately submerged in RNAlater (Life Technologies). Samples were stored at 4°C for 24 hours, after which supernatant was removed and samples were transferred to −80°C. Frozen tumors were dissociated in TRIzol (Life Technologies) using a TissueRuptor (QIAGEN). RNA was extracted using chloroform and purified on RNeasy MinElute columns (QIAGEN). RNA integrity was confirmed on the Bioanalyzer (Agilent Technologies). Library preparation and sequencing (50–base pair single-end) were performed by Australian Genome Research Facility, using Illumina HiSeq standard protocols.