Single-cell RNA-Seq analysis and validation strategy. (A-B) Cryopreserved cells from dissociated FL tumor biopsies from 6 patients, 3 PBMC donors, and 2 healthy tonsil donors (A), were measured by droplet-based scRNA-Seq, capturing an average of 1951 to 8560 cells per sample (B). Low-quality cells and technical artifacts were identified algorithmically and discarded. (C) A portion of the same aliquots of each sample was measured by multicolor flow cytometry (FACS) to validate immune and tumor subset frequencies observed by scRNA-Seq. (D-E) Published immune signatures for 8 purified immune subsets (D) were used to assign lineages (E) to each cell based on gene expression. (F) Tumor-infiltrating normal and tumor-derived B cells had distinct gene expression profiles. Tumor-specific candidate genes were characterized by FACS on additional cryopreserved aliquots of the tumor biopsies, yielding tumor-specific expression at the protein level. (G) The scRNA-Seq data of tumor-infiltrating T cells revealed the landscape of immune checkpoint gene coexpression.