title: Single-cell RNA-seq and CITE-seq of autochthonous KP and KPN mouse tumors
design: Single-cell RNA-seq and CITE-seq of autochthonous KP and KPN mouse tumors
library_construction_protocol: Two autochthonous mouse tumours with different genotypic mutations (KP: KrasG12D/+, Trp53fl/fl; KPN: KrasG12D/+, Trp53fl/fl, Rosa26N1icd/+) were harvested and stored in the cryopreserved material (CryoStore10, STEMCELL Technologies) before use. Tissue samples were enzymatically dissociated using a digestion mix prepared fresh by combining Collagenase D (250 µg/mL, Sigma-Aldrich Cat. 11088866001), Dispase II (800 µg/mL, Thermo Scientific Cat. 17105041), and DNase I (100 µg/mL, Roche Cat. 10104159001) in RPMI medium. Frozen tumor tissues were thawed at room temperature and washed with RPMI to remove cryoprotectant. Following media removal, 4 mL of digestion mix was added per sample. Samples were incubated at 37°C in a benchtop Thermomixer C (Eppendorf) with shaking at 500-600 RPM for 20 minutes. After incubation, tissues were mechanically disrupted by pipetting with widened-bore tips, and the supernatant containing dissociated cells was collected and incubated on ice. Fresh digestion mix was added to remaining tissue and incubated for additional 10-minute intervals with intermittent mechanical disruption, repeating until complete digestion. Digestion supernatants from the same sample were pooled, filtered through a 70 µm strainer, and centrifuged at 400g for 10 minutes at 4°C. Cell suspensions from two tumor samples were individually labeled with TotalSeq™-C0308 anti-mouse Hashtag 8 antibody and TotalSeq™-C0314 anti-mouse Hashtag 14 antibody (both from BioLegend) and combined for staining with TotalSeq™-C Mouse Universal Cocktail, V1.0 (BioLegend). To enrich stromal cell populations and enable compartment-specific analysis, dissociated single cells were FACS-sorted into epithelial (Epcam⁺Cd45⁻), immune (CD45⁺EpCAM⁻), and stromal (EpCAM⁻CD45⁻) populations for subsequent downstream analysis (Flow antibodies: BD Biosciences, Cat. 740281; BioLegend, Cat. 147707). Mouse tumours were induced at the age of 6-8 weeks old with intraperitoneal injection of tamoxifen at 80mg/kg. NA Single-cell RNA sequencing (scRNA-seq) libraries were prepared using the Chromium Single Cell 5' v3 Reagent Kits (10x Genomics) according to the manufacturer's protocol. 