characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-11-18T17:00:06Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-12-31T00:58:33Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110695929
characteristics.characteristic: External Id
characteristics.text: IRB Barcelona / BIST
characteristics.characteristic: INSDC center name
characteristics.text: 2022-11-18T17:00:06Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-12-31T00:58:33Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12793476
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11981:BYLQLGNG
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: neoplastic cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: 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.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: metastatic colorectal cancer
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: GFP+; CD45-
characteristics.characteristic: immunophenotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: liver micrometastasis
characteristics.characteristic: organism part
characteristics.text: pass
characteristics.characteristic: post analysis well quality
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: OK
characteristics.characteristic: single cell well quality
characteristics.text: C57BL/6J
characteristics.characteristic: strain
characteristics.text: BYLQLGNG
characteristics.characteristic: title