a, Schematic overview of the study design (see detailed descriptions and notations in the Methods). Two reference cell lines (sample A, HCC1395; sample B, HCC1395BL) were used to generate scRNA-seq data across four platforms (10x Genomics, Fluidigm C1 HT, Fluidigm C1 and Takara Bio ICELL8) and four testing sites (LLU, NCI, FDA and TBU). At the LLU and NCI sites (10x), mixed single-cell captures and library constructions were also prepared with either 10% or 5% cancer cells spiked into the B lymphocytes. At the NCI site, single-cell captures and library constructions were also performed with two methanol-fixed cell mixtures (5% cancer cells spiked into B lymphocytes, termed fixed_1 and fixed_2). One set of 10x scRNA libraries from the NCI was also sequenced using a shorter modified sequencing method. BK RNA-seq data were also obtained from these cell lines, each in triplicate. See Methods for details about study design. b, For both the breast cancer cell line (sample A) and the B lymphocyte line (sample B) across 14 pairwise datasets, percentages are shown of reads that mapped to the exonic region (blue) or the non-exonic region (orange) or did not map to the human genome (gray). For UMI methods (10x), dark blue indicates the exonic reads with UMIs. c, Median number of genes detected per cell at different sequencing read depths.