Jan 23, 2026
Neuron dissociation protocol for single-cell sequencing applications
- Boxun Li1,
- Charles Gersbach1
- 1Duke University
- Gersbach Lab
Protocol Citation: Boxun Li, Charles Gersbach 2026. Neuron dissociation protocol for single-cell sequencing applications. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1or5g5r/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: January 16, 2026
Last Modified: January 23, 2026
Protocol Integer ID: 238825
Keywords: Neuron dissociation, single-cell sequencing applications, neuron dissociation protocol, methods for neuron dissociation, cell sequencing application, neuron dissociation, cell sequencing applications this protocol, sequencing application, cell
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Boxun Li in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for neuron dissociation in preparation for single-cell sequencing applications.
Materials
- 1x Dulbecco’s (D) PBS (DPBS) (no Ca2+/Mg2+, Thermo Fisher, 14190-144)
- Dissociation solution: Papain (>=20U/mL) in Accutase with 10µM ROCK inhibitor (ROCKi).
o Papain: Worthington, LK003178
o Accutase: Millipore, SCR005
o ROCK inhibitor: Stemcell Tech, 72304
- DNase I (Worthington, LK003170): Make stock aliquots at 12500U/mL.
o DNaseI should be thawed right before the experiment.
Neuronal Maturation Medium (same as in protocol 1.Ngn2 neuron differentiation)
- Resuspension Medium: 49mL Maturation Medium + 1mL DNaseI stock supplemented with 10µM ROCKi.
o Warm up
o Add DNaseI right before trituration after 40-m incubation
o Prepare 5mL per 10cm dish for trituration
- Maturation Medium + 1% BSA
o 45mL Maturation Medium + 5mL 10% BSA solution (Sigma-Aldrich, A1595)
Troubleshooting
Prepare reagents
Prepare dissociation solution (Accutase/papain/ROCKi) right before when cells are ready:
- 1mL/well dissociation solution is needed per well for 6-well plates. Scale accordingly to other vessels.
- Dissolve each vial of papain with 5mL warm Accutase.
- To every 5mL of Accutase/papain solution, add 5µL ROCKi.
Put an aliquot of enough Maturation Medium in water/bead bath to warm up.
Aspirate medium from neurons. Wash once with 1x DPBS. Aspirate DPBS.
Note: Use 1mL/well DPBS for 6-well plates. Scale accordingly for different vessels.
Gently add dissociation solution to neurons without disturbing it. Use the same amount as DPBS.
Incubate the neurons at 37 °C for 40 min.
- Near the end of the incubation, add DNaseI and ROCKi to warm Maturation Medium to make Resuspension Medium. 5mL is needed for each full 6-well plate or 10cm dish. Aliquot 5mL of Resuspension Medium to each 15mL conical tube for a number of tubes, determined by the number of samples.
At the end of the incubation, neurons should easily come off the cell culture vessel as a sheet by rocking the vessel. Very gently, using a 10mL serological pipette, transfer the cell sheet(s) to a 15mL tube already containing 5ml Resuspension Medium, leaving behind as much dissociation solution in the vessel as possible.
Gently triturate the content of each tube 50x using a P1000 pipette to dissociate cell sheets into single cells.
Note:
- First 10 passes need to be slow to avoid over-shredding the cells. It will feel easier after 10 passes.
- Sufficient dissociation is critical at this step. It is recommended to check the cell suspension under a microscope and if necessary, pipette the cell suspension up and down more until the majority becomes single cells. However, over-pipetting could result in poor viability.
Pool contents of tubes together after pipetting, if desired.
The protocol diverges here depending on the downstream application (flow cytometry or scRNA-seq)
For flow cytometry:
- Centrifuge cells at 300xg for 5 minutes.
- Resuspend cells in DPBS + 1% BSA.
- Cells are ready for flow cytometry.
For bulk ATAC-seq:
- Centrifuge cells at 300xg for 5 minutes.
- Resuspend cells in freeze media (90% KnockOut Serum Replacement (Gibco, 10828010) + 10% dimethyl sulfoxide (Sigma-Aldrich, D8418)).
- Freeze cells at -80C in Mr. Frosty Freezing Container (Thermo Scientific, 5100-0001).
- Transfer frozen vials to liquid nitrogen for storage until ready for ATAC-seq.
For scRNA-seq:
Centrifuge cells at 300xg for 5 minutes.
- Add 5mL/tube Resuspension Medium. Pipette triturate gently 20x using P1000.
- Centrifuge cells at 300xg for 5 minutes.
- Add 1mL/tube Maturation Medium + 1% BSA. Pipette triturate gently 20x using P1000. Transfer cell suspensions to 1.7mL Eppendorf tubes.
- Centrifuge cells at 300xg for 5 minutes with the table-top centrifuge, discard supernatants, and resuspend each pellet with 1mL Maturation Medium + 1% BSA with 20x gentle trituration by P1000 after each spin. Repeat this for a total of 3 times.
- After the second spin and resuspension, take 10µL to count each sample on a hemocytometer with Trypan blue.
- After the third spin, resuspend cell pellets thoroughly 30-50x by pipetting the cells gently up and down with P1000 in MM+1% BSA to a target concentration of 1500 cells/µL based on the previous count. It is expected that the actual concentration is lower than the target due to cell loss during the spin and straining steps below.
- Pass cells through 30 µm Pre-Separation Filter (Miltenyi Biotec, 130-041-407) into a 15mL conical tube.
- Count cells on a hemocytometer (2 replicates per sample) with Trypan blue. This will generate the final concentration to determine the loading volume onto the 10X Genomics chip.
- Proceed to cell loading for 10X Genomics 5’ HT v2 scRNA-seq assay.