Single-Cell Integrated Mutation and Expression

Integrated detection of somatic mutations, SNPs within regions of chromosomal deletion, and gene expression in the same single cell was performed on a Biomark HD instrument (Fluidigm) (Livak et al. 2013 (link); Vargas et al. 2016 (link)). cDNA was generated using reverse transcription master mix (100-6299, Fluidigm), and preamplification was performed with PreAmp master mix (100-5744, Fluidigm) and 10× preamplification primer mix (500 nM each primer). For gene expression, allele-specific mutation and SNP detection, qPCR was performed using 96.96 dynamic array IFCs (Fluidigm) as previously described (Livak et al. 2013 (link); Burger et al. 2016 (link)). Data were analyzed with the Fluidigm real-time PCR analysis software using the linear (derivative) baseline correction method and the auto (global) Ct threshold method. The C_q values determined were exported, and data were processed as the C_q threshold (set to 28) minus the experimental C_q values. The transformation yields values equivalent to a log2 transformation of sequencing read count. For more details on primer design, cDNA generation, and preamplification conditions, please see the Supplemental Methods.