D3 software

Gene expression and expressed SNVs were assessed by qPCR or SNP type PCR across single cells using a Biomark HD system (Fluidigm). cDNAs obtained from the C1 array for mRNA sequencing chip were subjected to specific target amplification following the manufacturer’s recommendations. For the gene expression qPCR, Delta Gene Assay (Fluidigm) with EvaGreen second generation dsDNA binding dye was performed for gene sets selected from the RS genes (Additional file 6). To compare correlations between RNA-seq and qPCR platforms for the selected 43 gene expression, mean fold change over median expression was calculated as in the previous study [33 (link)]. Validation of expressed SNVs at the RNA level was carried out using a SNP Type Assay (Fluidigm) with locus-specific primer sequences. Primers were designed using D3™ software (Fluidigm), and sequences are available in Additional file 6.

qPCR was performed with the DELTAgene assay (PN100-3035, Fluidigm) using cDNAs from 6 bulk and 185 single-cell samples. Primer sequences were designed using D3 software (Fluidigm) and are listed in Supplementary Table 6. Before comparison of qPCR and RNA-seq data, Ct values of 999 (=not detected) were replaced with ‘NA'. Ct values were negatively converted and −20 was set as the threshold value. These data represent the log₂ expression level for qPCR comparable to log₂(TPM+1) for RNA-seq. The inter-relations were assessed by Pearson's correlation, Spearman's rank order correlation and linear regression analysis.