Nov 17, 2025
Midbrain organoid dissociation for Single-cell transcriptome library preparation and sequencing
- Hariam Raji1,
- michela.deleidi 1
- 1Institute Imagine Paris (24 Bd du Montparnasse, 75015 Paris, France)
- Hariam Raji: Mechanisms and Therapy of Genetic Brain Diseases
- michela.deleidi : Mechanisms and Therapy of Genetic Brain Diseases
- Team Deleidi
Protocol Citation: Hariam Raji, michela.deleidi 2025. Midbrain organoid dissociation for Single-cell transcriptome library preparation and sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.261gek7odg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 05, 2025
Last Modified: November 17, 2025
Protocol Integer ID: 231606
Keywords: enzymatic dissociation of midbrain organoid, midbrain organoid dissociation, midbrain organoid, cell transcriptome library preparation, 10x genomics scrna, preparation of 10x genomics scrna, organoid, enzymatic dissociation, illumina novaseq platform, cell suspension, midbrain, seq library
Abstract
This protocol describes the enzymatic dissociation of midbrain organoids into single-cell suspensions, preparation of 10x Genomics scRNA-seq libraries, and high-depth sequencing on an Illumina NovaSeq platform.
Guidelines
iPSC-derived midbrain organoids were dissociated into single-cell suspensions using a Worthington Papain Dissociation System kit according to manufacturer’s instructions. Organoids were minced and incubated with 2.5 mL papain/DNAase solution on an orbital shaker for 30 min at 37 °C. Organoid suspensions were triturated using scalpels and 1 mL low-attachment pipette tips and then incubated on an orbital shaker for 20 min at 37 °C twice. After enzyme inactivation, the cell suspensions were filtered with a 40-µm cell strainer. The final cell suspension was centrifuged at 300×g for 7 min, and the cell pellet was resuspended in PBS-0.04% BSA at a final concentration of 1000 cells/µL, ensuring that more than 95% of the cells were viable. scRNA-Seq libraries were generated with the 10X Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 according to the manufacturer’s instructions. Libraries were pooled and subjected to paired-end sequencing on the Illumina NovaSeq X platform with a sequencing depth of 300 million reads per library.
Materials
Worthington Papain Dissociation System kit (Worthington Biochemical), 2.5 mL papain/DNAase solution, scalpel, 1 mL low-attachment pipette tips, 40-µm cell strainer, PBS-0.04% BSA, 10X Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (Cat. number 1000128, 10x Genomics), Illumina NovaSeq X platform (SP Flow Cell, Illumina)
Troubleshooting
Midbrain organoid dissociation for Single-cell transcriptome library preparation and sequencing
Dissociate iPSC-derived midbrain organoids into single-cell suspensions using a Worthington Papain Dissociation System kit according to manufacturer’s instructions.
Mince individual organoids using scalpels and incubate with 2.5 mL papain/DNAase solution on an orbital shaker for 30 min at 37 °C.
After 30 min at 37 °C, triturate organoid suspensions using 1 mL low-attachment pipette tips, then incubate on an orbital shaker for another 20 min at 37 °C.
After enzyme inactivation using the inhibition solution, filter the cell suspensions with a 40-µm cell strainer. Centrifuge the final cell suspension at 300×g for 7 min, and resuspend the cell pellet in PBS-0.04% BSA at a final concentration of 1000 cells/µL, ensuring that more than 95% of the cells are viable.
scRNA-Seq libraries were generated with the 10X Chromium Next GEM Single Cell 3’ Reagent
Kit v3.1 (Cat. number 1000128, 10x Genomics, Pleasanton, CA, USA) according to
the manufacturer’s instructions. Libraries were pooled and subjected to
paired-end sequencing on the Illumina NovaSeq X platform (SP Flow Cell,
Illumina) with a sequencing depth of 300 million reads per library.