Single-cell RNA sequencing of primary and metastatic melanoma lesions in mice
This experiment used single-cell RNA sequencing to characterize transcriptional heterogeneity and tumor-cell state dynamics in primary and metastatic lesions from a mouse B16 melanoma model. For each experimental group, small pieces of tumor tissues were collected from five mice and pooled to generate one sequencing sample. The pooled tumor tissues were dissociated into single-cell suspensions. Single-cell cDNA libraries were generated using the BD Rhapsody single-cell 3' whole transcriptome amplification (WTA) protocol. Briefly, single cells were washed, counted, loaded onto a cartridge, and lysed inside the wells, followed by mRNA capture on cell capture beads, reverse transcription, cDNA extension, amplification, and library construction according to the manufacturer's instructions. Libraries were sequenced on the Illumina NovaSeq X Plus system using 150 nt paired-end sequencing. Raw sequencing reads were processed to generate gene-barcode expression matrices. The resulting matrices were analyzed using Seurat v4.4.0 for quality control, normalization, dimensionality reduction, clustering, cell-type annotation, tumor-cell state annotation, SCENIC/AUCell regulon analysis, and pseudotime analysis.
This experiment used single-cell RNA sequencing to characterize transcriptional heterogeneity and tumor-cell state dynamics in primary and metastatic lesions from a mouse B16 melanoma model. For each experimental group, small pieces of tumor tissues were collected from five mice and pooled to generate one sequencing sample. The pooled tumor tissues were dissociated into single-cell suspensions. Single-cell cDNA libraries were generated using the BD Rhapsody single-cell 3' whole transcriptome amplification (WTA) protocol. Briefly, single cells were washed, counted, loaded onto a cartridge, and lysed inside the wells, followed by mRNA capture on cell capture beads, reverse transcription, cDNA extension, amplification, and library construction according to the manufacturer's instructions. Libraries were sequenced on the Illumina NovaSeq X Plus system using 150 nt paired-end sequencing. Raw sequencing reads were processed to generate gene-barcode expression matrices. The resulting matrices were analyzed using Seurat v4.4.0 for quality control, normalization, dimensionality reduction, clustering, cell-type annotation, tumor-cell state annotation, SCENIC/AUCell regulon analysis, and pseudotime analysis.