GSM4726799: KPPCN 2; Mus musculus; RNA-Seq
For single-cell sequencing, KPPC and KPPCN mice with advanced tumors, 3 of each genotype, were sacrificed 7-8 weeks of age. Single-cell suspensions were isolated from minced tumors using Miltenyi Biotec GentleMacs dissociator in gentleMACS C Tubes (#130-093-23) and mouse tumor tissue dissociation kit (#130-096-730) as per the manufacturer's instructions. Dead cells were removed by use of Dead Cell removal microbeads (#130-090-101), hematopoeitic cells were depleted by CD45 MicroBeads (#130-052-30). The Chromium controller was used to make single-cell droplet with GEM bead. Single-cell suspensions were converted to barcoded scRNA-seq libraries by using the Chromium Single Cell 3′ Library, Gel Bead & Multiplex Kit and Chip Kit V3 (10X Genomics, #PN-1000092) loading an estimated 6,000 cells per library and following the manufacturer's instructions. Samples were processed using kits pertaining to the V3 barcoding chemistry of 10x Genomics. For each replicate, all tumor samples were processed in parallel in the same thermal cycler. The final libraries were profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies) and quantified using the Qubit 2.0 (Thermal Fisher, #Q32851). Each single-cell RNA-seq library  was sequenced twice in two lanes of HiSeq 4000 (Illumina) to obtain single-end, 98 bp, approximately 500 million reads per library.