GSM9193473: Long Tx-Group 1, GEX; Mus musculus; RNA-Seq
Yummer 1.7 melanoma tumor tissue was digested in RPMI/2% FBS supplemented with 0.1 mg/mL collagenase (Sigma) and 1:1000 DNase I (Sigma) for 30 min at 37ºC and then mechanically dissociated. Dissociated tumors were passed through a 70 μM filter and washed in RPMI medium to create a single cell suspension. The cell suspension was subjected to fluorescence-activated cell sorting (FACS) for CD45 expression, yielding CD45+ (immune cell) and CD45- (tumor cell) populations. Post-sorting, the cells were recombined to enrich for immune cells using 20% CD45- and 80% CD45+ cells to achieve a final population of 100,000 cells. This enriched cell suspension was processed using the Chromium 5′ Single-Cell Gene Expression system (10X Genomics) to generate scRNA seq libraries following the manufacturer's protocol. Briefly, single-cell suspensions with ≥85% viability were incubated with a pool of oligonucleotide-conjugated antibodies (BioLegend TotalSeq™) for sample indexing. Labeled cells were loaded into a Chromium Next GEM Chip G to generate Gel Bead-In-Emulsions (GEMs), enabling barcoding of individual cells and transcripts. Reverse transcription, cDNA amplification, and separate library construction for gene expression and feature barcodes were performed as described in the 10x Genomics protocol. Libraries were quantified, pooled, and sequenced on an Illumina NovaSeq platform with recommended read configurations.