ERS10393551 SAMEA12788201 10090 Mus musculus Protocols: AKTP organoids were derived from genetically-modified mice bearing conditional alleles in homologues of four key human CRC mutations: Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl and Trp53fl/fl (designated A, K, T and P, respectively) and targeted gene recombination to intestinal stem cells by means of the Lgr5eGFP-creERT2 driver. Intra-caecum injections of mouse tumor organoids carrying Apc, K-ras, p53 and TGFBR2 driver alterations were used for the generation of primary tumors at 7 to 9 weeks of age. Mice were anesthetized with isoflurane and placed in dorsal recumbency. The abdomen was shaved and sterilized with providone-iodine surgical solution. A small midline incision - slightly to the left- was performed to open the skin and peritoneum and expose the abdominal area. We placed a sterile surgical drape on top of the abdomen with a circular hole above the incision and sprawled the caecum over the drape using cotton swabs and saline to keep it hydrated. After confirming the presence of a primary tumour, Kelly forceps were used to first knot the surgical suture into the caecum wall, in between the ileocecal junction and the primary tumour. This provided a grip for subsequent caecum ligation. After ligation, the apical caecum containing the primary tumour was excised and any remaining cecal tissue was trimmed. Primary tumours were chopped with razor blades. Subsequent enzymatic digestion was performed with 200 U/ml collagenase IV (Sigma Aldrich) in HBSS (Lenovo) for 30 min at 37 °C, in a shaking water bath or a gentleMACS Dissociator (Miltenyi Biotec). Digested tissue fragments were then filtered through 100- and 40-μm meshes, washed and treated for 5 minutes with ammonium chloride. 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). RNA from 2000 cells was extracted and retrotranscribed to cDNA as described previously From FACS analysis, single cells were sorted into 1.5 mL tubes (Eppendorf). Cell concentration was adjusted to approximately 1,000 cells/μl and approximately 8,300 sorted cells were used for 3' single-cell gene expression profiling. Cell partition into GEMs was carried out on the Chromium platform (10x Genomics), according to the manufacturer instructions for. Briefly, 5,000 cells were targeted for partitioning into GEMS and the reverse-transcribed cDNA was amplified using 12 cycles. The resulting cDNA was quality controlled using a high sensitivity DNA assay on the Bioanalyzer 2100 (Agilent) system. The purified libraries were quality controlled, quantified and used to prepare a 20 nM equimolar sequencing pool. ENA first public 2022-02-11 ENA last update 2022-02-11 External Id SAMEA12788201 INSDC center alias IRB Barcelona / BIST INSDC center name IRB Barcelona / BIST INSDC first public 2022-02-11T00:13:54Z INSDC last update 2022-02-11T00:13:54Z INSDC status public Submitter Id E-MTAB-11284:Sample 1 broker name ArrayExpress common name house mouse developmental stage adult genotype Lgr5eGFP-creERT2; Apcfl/fl; KrasLSL-G12D; Tgfbr2fl/fl; Trp53fl/fl growth condition injection of mouse tumor organoids grown until neoplasm formation organism part distal caecum sample name E-MTAB-11284:Sample 1 sampling site neoplasm strain C57BL/6J ERS10393552 SAMEA12788202 10090 Mus musculus Protocols: AKTP organoids were derived from genetically-modified mice bearing conditional alleles in homologues of four key human CRC mutations: Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl and Trp53fl/fl (designated A, K, T and P, respectively) and targeted gene recombination to intestinal stem cells by means of the Lgr5eGFP-creERT2 driver. Intra-caecum injections of mouse tumor organoids carrying Apc, K-ras, p53 and TGFBR2 driver alterations were used for the generation of primary tumors at 7 to 9 weeks of age. Mice were anesthetized with isoflurane and placed in dorsal recumbency. The abdomen was shaved and sterilized with providone-iodine surgical solution. A small midline incision - slightly to the left- was performed to open the skin and peritoneum and expose the abdominal area. We placed a sterile surgical drape on top of the abdomen with a circular hole above the incision and sprawled the caecum over the drape using cotton swabs and saline to keep it hydrated. After confirming the presence of a primary tumour, Kelly forceps were used to first knot the surgical suture into the caecum wall, in between the ileocecal junction and the primary tumour. This provided a grip for subsequent caecum ligation. After ligation, the apical caecum containing the primary tumour was excised and any remaining cecal tissue was trimmed. Primary tumours were chopped with razor blades. Subsequent enzymatic digestion was performed with 200 U/ml collagenase IV (Sigma Aldrich) in HBSS (Lenovo) for 30 min at 37 °C, in a shaking water bath or a gentleMACS Dissociator (Miltenyi Biotec). Digested tissue fragments were then filtered through 100- and 40-μm meshes, washed and treated for 5 minutes with ammonium chloride. 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). RNA from 2000 cells was extracted and retrotranscribed to cDNA as described previously From FACS analysis, single cells were sorted into 1.5 mL tubes (Eppendorf). Cell concentration was adjusted to approximately 1,000 cells/μl and approximately 8,300 sorted cells were used for 3' single-cell gene expression profiling. Cell partition into GEMs was carried out on the Chromium platform (10x Genomics), according to the manufacturer instructions for. Briefly, 5,000 cells were targeted for partitioning into GEMS and the reverse-transcribed cDNA was amplified using 12 cycles. The resulting cDNA was quality controlled using a high sensitivity DNA assay on the Bioanalyzer 2100 (Agilent) system. The purified libraries were quality controlled, quantified and used to prepare a 20 nM equimolar sequencing pool. ENA first public 2022-02-11 ENA last update 2022-02-11 External Id SAMEA12788202 INSDC center alias IRB Barcelona / BIST INSDC center name IRB Barcelona / BIST INSDC first public 2022-02-11T00:13:54Z INSDC last update 2022-02-11T00:13:54Z INSDC status public Submitter Id E-MTAB-11284:Sample 2 broker name ArrayExpress common name house mouse developmental stage adult genotype Lgr5eGFP-creERT2; Apcfl/fl; KrasLSL-G12D; Tgfbr2fl/fl; Trp53fl/fl growth condition injection of mouse tumor organoids grown until neoplasm formation organism part distal caecum sample name E-MTAB-11284:Sample 2 sampling site neoplasm strain C57BL/6J ERS10393553 SAMEA12788203 10090 Mus musculus Protocols: AKTP organoids were derived from genetically-modified mice bearing conditional alleles in homologues of four key human CRC mutations: Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl and Trp53fl/fl (designated A, K, T and P, respectively) and targeted gene recombination to intestinal stem cells by means of the Lgr5eGFP-creERT2 driver. Intra-caecum injections of mouse tumor organoids carrying Apc, K-ras, p53 and TGFBR2 driver alterations were used for the generation of primary tumors at 7 to 9 weeks of age. Mice were anesthetized with isoflurane and placed in dorsal recumbency. The abdomen was shaved and sterilized with providone-iodine surgical solution. A small midline incision - slightly to the left- was performed to open the skin and peritoneum and expose the abdominal area. We placed a sterile surgical drape on top of the abdomen with a circular hole above the incision and sprawled the caecum over the drape using cotton swabs and saline to keep it hydrated. After confirming the presence of a primary tumour, Kelly forceps were used to first knot the surgical suture into the caecum wall, in between the ileocecal junction and the primary tumour. This provided a grip for subsequent caecum ligation. After ligation, the apical caecum containing the primary tumour was excised and any remaining cecal tissue was trimmed. Primary tumours were chopped with razor blades. Subsequent enzymatic digestion was performed with 200 U/ml collagenase IV (Sigma Aldrich) in HBSS (Lenovo) for 30 min at 37 °C, in a shaking water bath or a gentleMACS Dissociator (Miltenyi Biotec). Digested tissue fragments were then filtered through 100- and 40-μm meshes, washed and treated for 5 minutes with ammonium chloride. 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). RNA from 2000 cells was extracted and retrotranscribed to cDNA as described previously From FACS analysis, single cells were sorted into 1.5 mL tubes (Eppendorf). Cell concentration was adjusted to approximately 1,000 cells/μl and approximately 8,300 sorted cells were used for 3' single-cell gene expression profiling. Cell partition into GEMs was carried out on the Chromium platform (10x Genomics), according to the manufacturer instructions for. Briefly, 5,000 cells were targeted for partitioning into GEMS and the reverse-transcribed cDNA was amplified using 12 cycles. The resulting cDNA was quality controlled using a high sensitivity DNA assay on the Bioanalyzer 2100 (Agilent) system. The purified libraries were quality controlled, quantified and used to prepare a 20 nM equimolar sequencing pool. ENA first public 2022-02-11 ENA last update 2022-02-11 External Id SAMEA12788203 INSDC center alias IRB Barcelona / BIST INSDC center name IRB Barcelona / BIST INSDC first public 2022-02-11T00:13:54Z INSDC last update 2022-02-11T00:13:54Z INSDC status public Submitter Id E-MTAB-11284:Sample 3 broker name ArrayExpress common name house mouse developmental stage adult genotype Lgr5eGFP-creERT2; Apcfl/fl; KrasLSL-G12D; Tgfbr2fl/fl; Trp53fl/fl growth condition injection of mouse tumor organoids grown until neoplasm formation organism part distal caecum sample name E-MTAB-11284:Sample 3 sampling site neoplasm strain C57BL/6J ERS10393554 SAMEA12788204 10090 Mus musculus Protocols: AKTP organoids were derived from genetically-modified mice bearing conditional alleles in homologues of four key human CRC mutations: Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl and Trp53fl/fl (designated A, K, T and P, respectively) and targeted gene recombination to intestinal stem cells by means of the Lgr5eGFP-creERT2 driver. Intra-caecum injections of mouse tumor organoids carrying Apc, K-ras, p53 and TGFBR2 driver alterations were used for the generation of primary tumors at 7 to 9 weeks of age. Mice were anesthetized with isoflurane and placed in dorsal recumbency. The abdomen was shaved and sterilized with providone-iodine surgical solution. A small midline incision - slightly to the left- was performed to open the skin and peritoneum and expose the abdominal area. We placed a sterile surgical drape on top of the abdomen with a circular hole above the incision and sprawled the caecum over the drape using cotton swabs and saline to keep it hydrated. After confirming the presence of a primary tumour, Kelly forceps were used to first knot the surgical suture into the caecum wall, in between the ileocecal junction and the primary tumour. This provided a grip for subsequent caecum ligation. After ligation, the apical caecum containing the primary tumour was excised and any remaining cecal tissue was trimmed. Primary tumours were chopped with razor blades. Subsequent enzymatic digestion was performed with 200 U/ml collagenase IV (Sigma Aldrich) in HBSS (Lenovo) for 30 min at 37 °C, in a shaking water bath or a gentleMACS Dissociator (Miltenyi Biotec). Digested tissue fragments were then filtered through 100- and 40-μm meshes, washed and treated for 5 minutes with ammonium chloride. 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). RNA from 2000 cells was extracted and retrotranscribed to cDNA as described previously From FACS analysis, single cells were sorted into 1.5 mL tubes (Eppendorf). Cell concentration was adjusted to approximately 1,000 cells/μl and approximately 8,300 sorted cells were used for 3' single-cell gene expression profiling. Cell partition into GEMs was carried out on the Chromium platform (10x Genomics), according to the manufacturer instructions for. Briefly, 5,000 cells were targeted for partitioning into GEMS and the reverse-transcribed cDNA was amplified using 12 cycles. The resulting cDNA was quality controlled using a high sensitivity DNA assay on the Bioanalyzer 2100 (Agilent) system. The purified libraries were quality controlled, quantified and used to prepare a 20 nM equimolar sequencing pool. ENA first public 2022-02-11 ENA last update 2022-02-11 External Id SAMEA12788204 INSDC center alias IRB Barcelona / BIST INSDC center name IRB Barcelona / BIST INSDC first public 2022-02-11T00:13:54Z INSDC last update 2022-02-11T00:13:54Z INSDC status public Submitter Id E-MTAB-11284:Sample 4 broker name ArrayExpress common name house mouse developmental stage adult genotype Lgr5eGFP-creERT2; Apcfl/fl; KrasLSL-G12D; Tgfbr2fl/fl; Trp53fl/fl growth condition injection of mouse tumor organoids grown until neoplasm formation organism part distal caecum sample name E-MTAB-11284:Sample 4 sampling site neoplasm strain C57BL/6J