GSM7427843: KPCA.A, omental tumor, scRNAseq; Mus musculus; RNA-Seq
Omental tumors (KPCA.A, KPCA.B, KPCA.C) were freshly collected from C57Bl6J mice and dissociated by incubating tumor pieces with a digestion buffer (DMEM 10% supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) at 37 °C for 30 min. The resulting mixture was transferred into a dissociation tube and processed using the MacMachine device to initiate the dissociation process. Cells were then filtered through a 70 µm strainer, washed, and resuspended in PBS. Single-cell RNA sequencing (scRNAseq) was performed on samples with >70% cell viability. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, tumor cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.