title: WT1_folfiri_2days
organism: Mus musculus
description: Protocols: AKP (Apc KO, KrasG12D and P53 KO) WT and KO organoids (Mouse Tumoral Organoids) were treated for 2d or 5d in FOLFIRI media. Folfiri was removed after 3 days, followed by addition of fresh fully-supplemented control media for two extra days (5d after FOLFIRI timepoint). Cells were sorted based on the expression of DAPI (Dapi negative to sort live cells). Only one KO (KO2) could be recovered in the 5d timepoint Organoids were culture as described in Tauriello et al. Nature 2018. AKP organoids were treated with FOLFIRI (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. Non multiplexed samples (Parental_folfiri_5days, WT1_folfiri_5days, KO2_folfiri_5days) Cell concentration and viability were verified by counting with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, S.A) after staining cells with Trypan blue.  Cells were partitioned into Gel Bead In Emulsions with a Target Cell Recovery of 6,000 total cells per sample. cDNA sequencing libraries were prepared using the Chromium Next GEM Single Cell 3ʹ v3.1 kit (10X Genomics) following manufacturer's instructions. Briefly, after GEM-RT clean up, cDNA was amplified during 13 cycles and cDNA QC and quantification were performed on a Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies). 100 ug of cDNA were used for library preparation. Multiplexed samples (all other) 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). Non multiplexed samples (Parental_folfiri_5days, WT1_folfiri_5days, KO2_folfiri_5days) Libraries were indexed by PCR using the PN-1000215 Dual Index Kit TT Set A. Size distribution and concentration of 3' cDNA libraries were verified on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies). Multiplexed samples (all other) 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: strain
attributes.value: C57BL/6J
attributes.tag: disease
attributes.value: colorectal carcinoma
attributes.tag: individual
attributes.value: parental clone 2
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: colorectum
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: ApcLoxP; Lgr5-EGFP-IRES-creERT2; LSL-K-rasG12D; p53LoxP; Tgfbr2LoxP
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-08
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-08
attributes.tag: growth condition
attributes.value: tumour organoid
attributes.tag: age
attributes.value: 2
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-08
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-08