characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-12-01T00:19:39Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-12-01T00:19:39Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA10536025
characteristics: External Id
characteristics: Max Planck Institute for Biology of Ageing
characteristics: INSDC center name
characteristics: 2022-12-01T00:19:39Z
characteristics: INSDC first public
characteristics: 2022-12-01T00:19:39Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS8187888
characteristics: SRA accession
characteristics: E-MTAB-11097:Old_organoids
characteristics: Submitter Id
characteristics: ArrayExpress
characteristics: broker name
characteristics: liver organoid cells
characteristics: cell type
characteristics: house mouse
characteristics: common name
characteristics: Protocols: For nuclei extraction, organoids were pooled from three different wells of mixed organoid dome cultures (young n>2, old n>3). After breaking the domes with mechanical dissociation, organoids were dounce homogenised in 1 ml EZ-buffer (SIGMA) (20 strokes with loose and a tight pestle, respectively) and spun 5 min at 300 g. The organoids were incubated on ice for 10 min in EZ-buffer supplemented with DNAseI NEB M0303S (4 units/ml) and 1X DNAseI buffer. Equal volume of EZ-buffer was added, the organoids were spun 5 min at 500 g and incubated again 10 min on ice in EZ-buffer supplemented with DNAseI NEB M0303S (8 units/ml) and 1X DNAseI buffer. Equal volume of EZ-buffer was added, the organoids were spun 5 min at 500 g, resuspended in NSB (1087.5 µl 1XPBS, 5.5 µl 2% BSA, 1.5 µl RNase Inhibitor) and filtered through a 0.22 µm strainer and resuspended again to reach 300,000 nuclei/ml for DroNc-seq. For the DroNc-seq libraries 155,000 Macosko V+ beads (86) were resuspended in 250 µl of cold Lysis buffer (6% w/v Ficoll PM-400, 0.2% Sarkosyl, 20mM EDTA, 200mM Tris pH 7.5, 50 mM DTT). Beads, emulsion oil and 75,000 nuclei in NSB (1087.5 µl 1X PBS, 5.5 µl 2% BSA, 1.5 µl RNase Inhibitor) were loaded on a Nadia Dolomite Bio instrument and run according to the manufacturer’s instructions. After emulsion breakage with 6X SSC/PFO (perfluorooctanol), the beads were washed with 6X SSC, reverse transcribed with TSO- Macosko primer and treated with ExoI. 2000 BEADS in 20 replicates were used for PCR with SMART PCR primer for 12 cycles. cDNA was purified with Ampure XP beads and run on a 5000 Bioanalyser High Sensitivity Chip. 600 pg of DNA in 6 replicates was tagmented with the Nextera XT kit and amplified by PCR using a 10 µM New-P5-SMART PCT hybrid oligo and a 10 µM Nextera N70 indexed oligo for 12 cycles. The DNA was purified with AMPpure XP beads and run on a 1000 Bioanalyser High Sensitivity chip.
characteristics: description
characteristics: adult
characteristics: developmental stage
characteristics: wild type genotype
characteristics: genotype
characteristics: organoid culture
characteristics: growth condition
characteristics: Mus musculus
characteristics: organism
characteristics: Mus musculus
characteristics: organism
characteristics: liver
characteristics: organism part
characteristics: Mus musculus
characteristics: scientific_name
characteristics: male
characteristics: sex
characteristics: C57BL/6N
characteristics: strain
characteristics: 18 months
characteristics: month
characteristics: time
characteristics: Old_organoids
characteristics: title