RNAseq of low-pigmented and high-pigmented cells from mouse B16 melanoma cell lines (experiment 1)

Functional variation due to underlying genetic and phenotypic heterogeneity poses a major challenge to long-term melanoma management. Melanin pigment production is the hallmark of normal melanocytic differentiation but varies significantly between and within melanoma tumours. Detailed assessment of the functional consequences of pigment heterogeneity in viable melanoma samples has been experimentally challenging. Here, we developed a novel method to prospectively separate melanoma cell populations based on the presence of intracellular melanin using fluorescence-activated cell sorting (FACS). We find that nearly all pigmented patient melanomas and melanoma cell lines comprise mixtures of cells ranging from very high to low or undetectable pigment content. Using viable sorted cells, we interrogated the functional characteristics of low pigmented cells (LPC) and high pigmented cells (HPC) in the B16-F10 mouse melanoma cell line. Overall design: The B16 mouse melanoma cell lines were cultured and separated into low pigmented cells (LPC) and high pigmented cells (HPC) based on their melanin pigment content by fluorescence-activated cell sorting (FACS), exploiting the light scattering properties of intracellular melanin. RNA sequencing was performed on sorted cells with 3x replicates for each group.