description: Protocols: AKP (Apc KO, KrasG12D and P53 KO) and APS (Apc KO, P53 and Smad4KO) mouse organoids were treated with 4OH-Tamoxifen for 16 hours before the addition of FOLFIRI. Cells were sorted right after Tamoxifen induction (after chemotherapy), and right after FOLFIRI treatment (sorted by their Tomato expression). 30 days after removal of folfiri, cells were recovered from folfiri treatment, and after a passage were sorted based on their tomato expression. Organoids were culture as described in Tauriello et al. Nature 2018. Organoids were grown for 3 days in normal organoid stem cell media before chemotherapy treatment began APS and AKP organoids were induced for 16h with 4-OH Tamoxifen (1 μM Sigma-Aldrich/Merck, H7904). FOLFIRI was a combination of 5FU (50μg/μl) and SN38 (100nM, MedChem HY-13704). After 3 days of treatment, drugs were removed, followed by addition of fresh fully-supplemented control media. Sample multiplexing was performed following the Cell Multiplexing Oligo Labeling for Single Cell RNA Sequencing Protocol (10x Genomics). Briefly, cells were centrifuged at 500x g at room temperature with a swinging-bucket rotor, resuspended in 100 ul of Cell Multiplexing Oligo (3' CellPlex Kit, 10x Genomics) and incubated at RT for 5 mins. Cells were washed 3 times with cold 1X PBS (Thermo Fisher Scientific) supplemented with 1% BSA (MACS Miltenyi), all centrifugations being performed at 500x g at 4C. Cells were finally resuspended in an appropriate volume of 1X PBS-0.05% BSA in order to obtain a final cell concentration of approximatively 1000 cells/ul and counted using a TC20™ Automated Cell Counter (Bio-Rad Laboratories, S.A). Samples were mixed with a 50:50 ratio and filtered with a 40 µm strainer to remove eventual clumps and the resulting pools were checked for final cell number and viability before loading onto the Chromium. Cellplex samples were partitioned into 3' Gel Bead Emulsions with a Target Cell Recovery between 6.000 and 20.000 cells (corresponding to 3.000-5.000 cells per sample within each plex). Libraries were prepared following 10x Genomics Single Cell 3' mRNA kit protocol with Feature Barcode technology for Cell Multiplexing. Briefly, after GEM-RT clean up, cDNA from poly-adenylated mRNA and barcoded DNA from the CMO Feature Barcode were amplified via PCR according to the Target Recovery cell number. A SPRI selection clean-up was done in order to separate the amplified cDNA molecules for 3' Gene Expression and the CMO-derived cDNA. 100 ng of mRNA-derived cDNA were used for GEX library construction while 5 µl of CMO-derived cDNA were used to amplify the corresponding Cellplex library. Size distribution and library concentration were determined using a Bioanalyzer High Sensitivity chip (Agilent Technologies)
attributes.tag: ENA first public
attributes.value: 2022-05-13
attributes.tag: ENA last update
attributes.value: 2022-05-13
attributes.tag: External Id
attributes.value: SAMEA14382923
attributes.tag: INSDC center alias
attributes.value: IRB Barcelona - BIST
attributes.tag: INSDC center name
attributes.value: IRB Barcelona - BIST
attributes.tag: INSDC first public
attributes.value: 2022-05-13T12:34:03Z
attributes.tag: INSDC last update
attributes.value: 2022-05-13T12:34:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11145:APS5_folfiripos_tomneg
attributes.tag: age
attributes.value: 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: colorectal adenoma
attributes.tag: fraction
attributes.value: no tomato expression
attributes.tag: genetic modification
attributes.value: Apc KO, P53 and Smad4KO
attributes.tag: genotype
attributes.value: APCMin/+ Mex3a CreErt2/+
attributes.tag: growth condition
attributes.value: tumour organoid
attributes.tag: organism part
attributes.value: colorectum
attributes.tag: sample name
attributes.value: E-MTAB-11145:APS5_folfiripos_tomneg
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6J