title: KNFODEMQ
organism: Mus musculus
description: Protocols: MTOs were established from primary tumors arising in genetically engineered mouse models bearing mutations in Apc, Kras, and p53 (AKP mouse model, Apc fl/fl, KrasG12D and Trp53 fl/fl). We analyzed by scRNAseq micro-dissected liver metastases generated by implantation of AKP MTOs in the caecum of c57BL/6 mice. The PGK is driven by a constitutive PGK promoter and therefore expressed in all tumor cells. GFP was used thus to separate tumor cells from the rest of healthy cells. For isolation of low numbers of tumour cells from mice without visible metastases, whole livers were thoroughly minced with razor blades. After an initial 30-minute digestion with 200 U/ml collagenase IV, samples were filtered through 100 μm meshes. Although most cells flowed through, a small fraction of the samples - highly enriched for tumour cells - was retained in the filter (Supplementary Figure 5c). Filters were then laid into a 6-well plate and covered with HBSS containing collagenase IV, Dispase II and DNase I. After re-digestion in a water bath for 30 minutes al 37 °C, most cells now seeped through the filters. Single-cell preparations were first blocked with anti-CD16/32 (clone 93; eBioscience) and then stained with APC anti-Epcam (118214; Biolegend) and BV605 anti-CD45 (103155, Biolegend) antibodies. Finally, cells were resuspended in HBSS with 0.5% FBS and DAPI (Sigma Aldrich). Freshly harvested GFP+ single cells were sorted into 96-well plates containing 2.5 µl of the Reaction buffer (1x Lysis Buffer, RNase Inhibitor 1U/µl). Full-length single-cell RNA-seq libraries were prepared using the SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing (Takara Bio). All reactions were downscaled to one quarter of the original protocol and performed following thermal cycling manufacturer's conditions.