Protocol for mechanical nuclei dissociation and isolation from frozen tissue and organoids for single-nucleus RNA sequencing

Summary

Preserving RNA integrity in complex tissues such as the brain is challenging. Here, we present a protocol for isolating intact nuclei from frozen tissue and organoids for single-nucleus RNA sequencing (snRNA-seq). We describe steps for mechanical dissociation, using the GentleMACS Dissociator, serial filtration, and magnetic bead-based nuclei isolation. We then detail nuclei quality control and counting. This approach yields pure single-nucleus suspensions suitable for downstream fixation, cryopreservation, or library preparation across diverse tissue types.

For complete details on the use and execution of this protocol, please refer to Guillon et al.¹

Graphical abstract

Highlights

• •Mechanical dissociation of frozen tissue and organoids using GentleMACS
• •Serial filtration and debris removal to obtain single-nucleus suspensions
• •Magnetic bead-based isolation and purification of nuclei
• •Nuclei counting and quality control before downstream library preparation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Preserving RNA integrity in complex tissues such as the brain is challenging. Here, we present a protocol for isolating intact nuclei from frozen tissue and organoids for single-nucleus RNA sequencing (snRNA-seq). We describe steps for mechanical dissociation, using the GentleMACS Dissociator, serial filtration, and magnetic bead-based nuclei isolation. We then detail nuclei quality control and counting. This approach yields pure single-nucleus suspensions suitable for downstream fixation, cryopreservation, or library preparation across diverse tissue types.

Before you begin

This protocol describes the isolation of intact nuclei from frozen tissue and organoids for single-nucleus RNA sequencing (snRNA-seq). By isolating nuclei rather than whole cells, snRNA-seq bypasses the difficulty of preserving RNA integrity in complex or frozen samples. The protocol has been established and validated on snap-frozen brain tissue and cultured cerebral organoids, and is broadly applicable to other tissue types with minor adjustments.

Tissue must be snap-frozen in liquid nitrogen or on dry ice immediately after collection and stored at −80°C. Slow freezing is not compatible with this protocol, as ice-crystal formation damages nuclei (see limitations). For organoids, the protocol has been validated from 6–7 days to up 4 months-old of differentiation; the optimal stage may vary with organoid type. The protocol is intended for unfixed tissue only—do not use already-fixed material.

Because RNases rapidly degrade RNA and compromise nuclei quality, decontaminate all surfaces, pipettes, and consumables with an RNase-removal reagent before starting, and keep all buffers on ice throughout. Dissociation and isolation require the GentleMACS Dissociator and the MACS magnetic separation system (Miltenyi Biotec); confirm both are available before beginning.

Innovation

Single-nucleus RNA sequencing (snRNA-seq) is often the only viable approach for transcriptomic profiling of frozen or hard-to-dissociate tissues, where intact cells cannot be recovered. Most established workflows rely on enzymatic digestion, which can introduce transcriptional stress artifacts and compromise nuclei integrity.² This protocol replaces enzymatic tissue digestion with a fully mechanical dissociation performed on the GentleMACS Dissociator, minimizing stress-induced artifacts while preserving RNA quality. DNase I is included in the buffers solely to digest free DNA released during lysis and to prevent nuclei aggregation; no protease or collagenase is used at any stage.

The protocol integrates two commercial reagent systems, Miltenyi nuclei extraction and Anti-Nucleus MicroBead isolation, into a single, streamlined, and reproducible pipeline, adapted and optimized beyond the manufacturers’ data sheets. Serial filtration through decreasing pore sizes, combined with magnetic bead-based capture, provides efficient, tunable debris removal and yields high-purity single-nucleus suspensions from challenging samples.

A further advance is its versatility: the same workflow accommodates both snap-frozen tissue and cultured organoids of different origins, with simple, tabulated adjustments of lysis buffer volume to match input amount. Built-in quality-control checkpoints (hemocytometer counting and morphological assessment by Trypan Blue and DAPI staining) allow users to monitor nuclei integrity at each stage, and an optional cryopreservation step enables banking of excess nuclei. The resulting suspension is directly compatible with downstream fixation and library-preparation chemistries (e.g., Parse Biosciences EVERCODE), making the protocol broadly transferable across experimental systems and sequencing platforms.

Buffer and reagent preparations

Timing: 20 min

This step prepares all buffers and reagents required before starting the dissociation. All buffers must be prepared fresh on the day of the experiment and kept on ice throughout the procedure.

• 1.Thaw the DNase I Solution slowly at 4°C, well in advance, as it has a long thaw time. Once thawed, keep DNase I and RNase Inhibitor on ice at all times.

CRITICAL: DNase I Solution has a long thawing time (1-2 h before beginning). Remove it from the freezer in advance and thaw slowly at 4°C before use.

• 2.Prepare a 2% BSA stock solution in D-PBS: For example, for 10ml of D-PBS, add 0.2g of Bovine serum albumin powder (see materials and equipment).
• 3.Prepare the Nuclei Lysis Buffer (NL Buffer) and Nuclei Separation Buffer (NS Buffer) fresh, following the recipes in Materials and equipment. Scale the NL Buffer volume to the tissue input. Keep all buffers on ice.

CRITICAL: Prepare all buffers freshly on the day of use and keep them on ice. Keep DNase I and RNase Inhibitor on ice at all times.

Workspace and consumables setup

Timing: 5 min

Set up the workspace before sample collection to minimize the time nuclei spend in the buffers.

• 4.Pre-cool the centrifuge(s) accepting 15 ml and 1.5 ml tubes to 4°C (see materials and equipment).
• 5.Fill an ice bucket with ice.
• 6.Equilibrate the Mr. Frosty freezing container to room temperature.
• 7.
  Label the tubes required for one dissociation:

  • a.
    1 × GentleMACS C Tube
  • b.
    2 × 15 mL conical tubes
  • c.
    4 × 1.5 mL tubes
• 8.Add 10 μL of Trypan Blue to two of the four 1.5 mL tubes. These tubes are used for nuclei counting at steps 12 and 18.

Note: If working with a tissue typically composed of a large number of cells (or more than 2 million cells), dilute the sample 1:10 in DNase/RNase-free water directly into the tube containing Trypan Blue prior to counting.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Mouse brain tissue C57BL/6 (hemisphere)	This study	N/A
Human ESC-derived cerebral organoids∗	This study	WIBR1
Chemicals, peptides, and recombinant proteins
Anti-Nucleus MicroBeads	Miltenyi Biotec	Cat#130-132-997
Bovine serum albumin (BSA)	Bio Basic	Cat# A0903
DAPI	Sigma-Aldrich	Cat#D9542
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat#D8418
DNase I Solution	STEMCELL Technologies	Cat#07900
Hydrochloric acid	Sigma-Aldrich	Cat#H1758
Nuclei Extraction Buffer	Miltenyi Biotec	Cat#130-128-024
Phosphate-buffered saline (D-PBS)	Sigma-Aldrich	Cat#P4474
RNase Inhibitor, Murine	New England Biolabs	Cat#M0314L
Trypan Blue stain, 0.4%	Thermo Fisher Scientific	Cat#T10282
UltraPure DNase/RNase-Free Distilled Water	Invitrogen	Cat#10977-015
Critical commercial assays
EVERCODE Nuclei Fixation v3	Parse Biosciences	Cat#ECFN3300
STEMdiff™ Cerebral Organoid Kit	STEMCELL Technologies	Cat#08570
Software and algorithms
BioRender	BioRender	https://www.biorender.com
Other
GentleMACS Dissociator	Miltenyi Biotec	Cat#130-093-235
GentleMACS C Tubes	Miltenyi Biotec	Cat#130-093-237
MACS MultiStand	Miltenyi Biotec	Cat#130-042-303
OctoMACS Separator	Miltenyi Biotec	Cat#130-042-109
MS Columns	Miltenyi Biotec	Cat#130-042-201
MACS SmartStrainers (100 μm)	Miltenyi Biotec	Cat#130-098-463
MACS SmartStrainers (70 μm)	Miltenyi Biotec	Cat#130-098-462
MACS SmartStrainers (30 μm)	Miltenyi Biotec	Cat#130-098-458
MACS Chill 15 Rack	Miltenyi Biotec	Cat#130-097-038
1.5 mL screw-cap microtubes	Diamed	Cat#DIATEC-2740
15 mL polypropylene conical tubes	FroggaBio	Cat#TB15-25
Disposable hemocytometer	Incyto	Cat#DHC-N01-5
Refrigerated centrifuge (15 mL and 1.5 mL rotors)	Avantor	Cat#76468-136; Cat#MP76533-878
Mr. Frosty freezing container	Sigma-Aldrich	Cat#CLS432001
25 mL reservoir	Corning	Cat#RES-V-25-S

^∗Organoids differentiated using STEMdiff™ Cerebral Organoid Kit (key resource table).

Materials and equipment

Cerebral organoids were generated with the STEMdiff Cerebral Organoid Kit (key resources table) by seeding 9000 hESCs per well in 96-well round-bottom ultra-low-attachment plates on day 0, and were processed between day 6 and 4 months of differentiation. Because cell content per organoid increases steeply with age, the number of organoids pooled per dissociation is adjusted to reach an input of approximately 1 × 10⁶ cells. The volumes below (4 mL NL Buffer, 8 mL NS Buffer) correspond to one such input; scale proportionally for other inputs.

Nuclei Lysis Buffer (NL Buffer)

Reagent	Final concentration	Amount
Nuclei Extraction Buffer (1×)	1×	3.972 mL
RNase Inhibitor, Murine (40 U/μL)	0.2 U/μL	20 μL
DNase I Solution (1 mg/mL)	2 μg/mL	8 μL
Total	N/A	4 mL

Final concentrations assume a 1 mg/mL DNase I stock and a 40 U/μL RNase Inhibitor stock; verify against your reagent lots before use. The final volume covers both pre-filling the C Tube (step 1) and the rinse (step 8).

Nuclei Separation Buffer (NS Buffer)

Reagent	Final concentration	Amount
D-PBS (1×)	1×	6.66 mL
BSA (stock solution 2%)	0.04%	160 μL
Nuclei Extraction Buffer (1×)	0.14×	1.12 mL
DNase I Solution (1 mg/mL)	5 μg/mL	40 μL
RNase Inhibitor, Murine (40 U/μL)	0.1 U/μL	20 μL
Total	N/A	8 mL

Final concentrations assume a 1 mg/mL DNase I stock and a 40 U/μL RNase Inhibitor stock; verify against your reagent lots before use.

Nuclei Lysis Buffer volume by tissue input for filling the C-Tube

Quantity or type of tissue	NL buffer volume (step 1)
Organoid (6-days to up 4 months-old)	2 mL
Tissue, up to 50 mg	1.5 mL
Tissue, 50–100 mg	3 mL
Tissue, 100–200 mg	6 mL
Mouse brain hemisphere	8 mL

The 4 mL recipe above covers one organoid/cultured-tissue dissociation (≈2 mL to pre-fill the C Tube plus ≈2 mL to rinse it); scale the recipe proportionally for larger inputs.

CRITICAL: Dimethyl sulfoxide (DMSO) and hydrochloric acid are hazardous. Handle them in a fume hood with appropriate personal protective equipment, and dispose of waste according to institutional guidelines.

Alternatives: The MACS Chill 15 Rack can be replaced by any pre-chilled tube rack kept on ice. The disposable hemocytometer can be replaced by a reusable Neubauer counting chamber; automated cell counters are not recommended at the filtration step because of the high debris content.

Step-by-step method details

Sample collection and mechanical dissociation

Timing: 10–15 min

Here, we describe steps for collecting frozen or cultured samples and mechanically dissociating them using the GentleMACS Dissociator to release nuclei while minimizing RNA degradation.

Collect the sample directly into ice-cold NL Buffer and mechanically disrupt it on the GentleMACS Dissociator to release nuclei. Work quickly to limit RNA degradation.

CRITICAL: Do not exceed 10 min between sample collection and the start of the dissociation program, to limit tissue degradation before the sample enters the buffer.

• 1.Pre-fill the GentleMACS C Tube with the appropriate volume of NL Buffer for the tissue type and keep it on ice.
• 2.
  Transfer the sample into the pre-filled C Tube:

  • a.
    For snap-frozen tissue: retrieve the tissue from −80°C storage and place it immediately into the pre-filled C Tube.

    Note: If the transfer time is more than 10 min, keep the sample on dry ice during the transfer.

  • b.
    For live or cultured tissue: using a 1000 μL wide-bore tip, collect the tissue in a small volume of medium into a 1.5 mL tube, then transfer the tissue without the medium into the pre-filled C Tube.
• 3.Close the C Tube by pressing the cap until it clicks, confirming a secure seal.
• 4.Place the C Tube upside down on the GentleMACS Dissociator and run the 4C_nuclei program (5 min 15 s) (Figure 1).
• 5.Immediately after the program, detach the C Tube and inspect the suspension; it should be turbid and homogeneous (Figure 1B, left), containing nuclei and cellular debris. Place the C Tube on ice.

CRITICAL: If macroscopic tissue fragments are still visible, see troubleshooting, problem 1.

Figure 1.

Mechanical dissociation and nuclei pelleting

(A) Step-by-step schematic representation of the protocol.

(B) Nuclei suspension before and after centrifugation; a white nuclei pellet is visible at the bottom of the tube after centrifugation, related to steps 9 and 13.

Filtration and debris removal to obtain a single-nucleus suspension

Timing: 15–20 min

Here, we describe steps for serial filtration and centrifugation to remove cellular debris and undissociated material and obtain a concentrated single-nucleus suspension.

Remove major cellular debris and undissociated material by serial filtration through SmartStrainers of decreasing pore size, yielding a single-nucleus suspension for magnetic isolation.

• 6.Place a labeled 15 mL tube on ice and fit the appropriate SmartStrainer on top (Figure 2A): 100 μm for snap-frozen tissue, or 70 μm for live or cultured tissue.

Note: For stability, seat the tube in a pre-chilled rack such as the MACS Chill 15 Rack.

• 7.Using a 1000 μL tip, pass the nuclei suspension through the strainer in one quick, continuous motion (≈1 s), keeping the tip just above the mesh without touching it (Figure 2B, left).
• 8.Add 2 mL of NL Buffer to the C Tube, close it, and invert it in all directions to rinse all internal surfaces. Briefly centrifuge at 100 × g for 1 min to collect the liquid, then pass it through the same strainer and discard the strainer.

Note: This rinse maximizes nuclei recovery from the C Tube.

• 9.Close the 15 mL tube and centrifuge at 300–400 × g for 5 min at 4°C.
• 10.Discard the supernatant and fully resuspend the pellet in 1 mL of NS Buffer.
• 11.Fit a 30 μm SmartStrainer on a new labeled 15 mL tube and pass the suspension through in one quick, continuous motion (≈1 s per stroke), keeping the tip in gentle contact with the mesh (Figure 2B, right).

Note: Flow may be slower through the 30 μm mesh. If it slows, move the tip to a different area of the mesh without lifting it away.

Note: For stability, seat the tube in a pre-chilled rack such as the MACS Chill 15 Rack.

• 12.Transfer 10 μL of the suspension (undiluted, or diluted 1:10 in DNase/RNase-free water for high-yield samples) into a prepared Trypan Blue tube and count the nuclei on a hemocytometer (Figure 3).

Note: A high nuclei concentration is typically expected for complex tissues such as human brain samples, where using more than 50 mg of starting material generally results in a high nuclear yield. Similarly, for organoids, high nuclear concentrations are expected once tissue complexity increases, such as after 1 month of differentiation.

Note: Do not use automated cell counters at this step. Substantial debris is expected here and is removed during the subsequent isolation steps (Figure 3A).

• 13.Centrifuge at 300–400 × g for 5 min at 4°C. A visible pellet should form (Figure 1B, right); if no pellet is visible, the nuclei concentration is low.
• 14.Discard the supernatant and resuspend the pellet in NS Buffer to ≈2,200 nuclei/μL (≈1 × 10⁶ nuclei in 450 μL).

Optional: Cryopreservation of excess nuclei. If the volume exceeds 450 μL, transfer the excess to a new labeled tube, add DMSO to 10% (v/v),³ mix, place in a Mr. Frosty container, and store at −80°C for up to 6 months.

Figure 2.

Workspace setup and serial filtration

(A) Tubes kept cold on a chilled MACS Chill 15 Rack during filtration.

(B) Filtration through a SmartStrainer into a 15 mL tube. Left (larger pore size): the pipette tip is held just above the mesh, without touching it. Right (30 μm): the pipette tip is in gentle contact with the mesh. Related to steps 6, 7, and 11.

Figure 3.

Hemocytometer counting and progressive debris removal (Trypan Blue)

Nuclei counted on a hemocytometer at successive stages of the protocol.

(A) High debris content immediately after dissociation.

(B–D) Progressively cleaner suspensions after magnetic isolation, ending in a clean single-nucleus suspension. Scale bars, 270 μm. Related to steps 12 and 17.

Magnetic isolation and purification of nuclei

Timing: 20–25 min

Here, we describe steps for labeling nuclei with Anti-Nucleus MicroBeads and purifying them by magnetic column separation to obtain a clean suspension for downstream processing.

Capture nuclei on Anti-Nucleus MicroBeads and purify them on a magnetic column. A 50 μL bead volume captures up to ≈1 × 10⁶ nuclei; scale the bead volume proportionally for higher yields.

• 15.Add 50 μL of Anti-Nucleus MicroBeads to 450 μL of NS, mix gently, and incubate for 15 min at 4°C.

Note: To keep the temperature stable during incubation, place the tube in an ice bucket inside the refrigerator.

Note: 50 μL is sufficient for up to ≈1 × 10⁶ nuclei. If more than ≈1 × 10⁶ nuclei are isolated, add an additional 50 μL for each extra ≈1 × 10⁶ nuclei, together with 450 μL of NS for every additional 50 μL.

CRITICAL: Do not exceed 15 min of incubation, as longer incubation may compromise nuclei integrity.

• 16.During the incubation, place the OctoMACS Separator on the MACS MultiStand and seat an MS Column (without the plunger; save it for elution, step 17d) on the separator, with a waste reservoir (e.g., a 25 mL reservoir) underneath. At 12 min, equilibrate the column with 1 mL of NS Buffer.
• 17.
  After the full 15 min incubation, isolate and elute the nuclei:

  • a.
    Using a non-wide-bore P1000 μl tip, load the suspension onto the center of the column while it remains on the magnet.
  • b.
    Once the suspension has fully passed through, wash with 1 mL of NS Buffer. Repeat for a total of three washes.
  • c.
    Remove the column from the magnet and place it on a labeled 1.5 mL tube.
  • d.
    Add 1 mL of NS Buffer and elute by firmly inserting the saved plunger; collect the purified single-nucleus suspension (Figure 3D).

Note: The beads retain nuclei within the column by magnetic interaction, while debris and unbound material flow through into the waste reservoir.

• 18.Proceed directly to fixation using the kit supplied with your library-preparation kit.

Note: During fixation, after a centrifugation step, the beads remain in suspension while the nuclei form a pellet, separating them naturally.

Optional: Before fixation, transfer 10 μL of the purified suspension (diluted 1:10 in DNase/RNase-free water if a more than 1 milion nuclei are expected) into a prepared Trypan Blue tube and count on a hemocytometer to estimate final yield and integrity. At this stage, the suspension should appear clean, with minimal debris compared with the earlier count (Figures 3D and 4).

Figure 4.

Nuclei integrity and morphology

(A and B) Nuclei in the counting chamber; arrowheads indicate intact single nuclei, debris, and aggregates.

(C) Representative transmitted-light (TRANS), DAPI, and overlay images. Intact nuclei are round with a smooth, well-defined nuclear envelope. Scale bars, 50 μm. Related to expected outcomes.

Expected outcomes

This protocol yields a purified single-nucleus suspension ready for fixation and downstream snRNA-seq library preparation. The expected concentration ranges from 1 × 10⁶ to 1 × 10⁸ nuclei/mL, depending on the tissue type and, for cultured tissues, the stage of differentiation. Throughout the procedure, cellular debris is progressively removed, producing an increasingly pure preparation (Figure 3).

Nuclei integrity, assessed by Trypan Blue exclusion, should be at least 90% at the end of the isolation. Intact nuclei appear as single, round structures with a smooth, well-defined nuclear envelope (Figure 4). This proportion of intact nuclei is required for efficient and accurate single-nucleus barcoding during library preparation. If not, refer to troubleshooting.Nuclei with irregular morphology (elongated, fragmented, or asymmetric shapes) or membrane irregularities may be damaged or apoptotic and indicate suboptimal tissue quality or processing. Nuclei aggregates must also be avoided, as clumped nuclei prevent accurate single-nucleus barcoding; if aggregates are present, the suspension is not a true single-nucleus suspension. If damaged nuclei or aggregates exceed 5–10%, see troubleshooting. A successful library prepared from these nuclei shows a single major peak at ≈418 bp by electrophoresis (Figure 5).

Figure 5.

Quality control of a sequencing library prepared from purified nuclei

(A and B) Electropherogram (fragment size versus normalized intensity) of a library generated from nuclei isolated with this protocol, showing a single major peak at ≈418 bp between the lower and upper markers. Related to expected outcomes.

(A) cDNA sublibrary.

(B) Ready-to-sequence library.

Quantification and statistical analysis

Nuclei concentration and viability are determined from hemocytometer counts after Trypan Blue staining. Concentration (nuclei/mL) = mean number of nuclei per corner square × dilution factor × 10⁴. Integrity (%) = (number of unstained nuclei/total number of nuclei) × 100. A preparation is considered suitable for downstream processing when at least 90–95% of nuclei are intact and unstained.

Limitations

This protocol has been established and validated on snap-frozen mouse brain tissue and cultured human cerebral organoids. Other tissue types are likely to require optimization, and the parameters most likely to need adjustment are indicated below.

Beyond the dissociation step itself, several parameters may require adjustment. The NL Buffer volume and the duration of the GentleMACS program should be matched to the tissue (steps 1 and 4); the 4C_nuclei program (≈5 min) may be insufficient for compact tissues such as muscle or adipose tissue. The strainer pore sizes and the number of filtration passes may need to be adapted for tissues with high connective or lipid content (steps 6, 7, and 11), and the DNase I concentration in both buffers may need to be increased for DNA-rich or highly necrotic samples, in which free DNA promotes nuclei aggregation. At the magnetic isolation stage, the bead volume and incubation time may require adjustment for tissues in which nuclei are captured less efficiently (step 15), and for fragile nuclei the centrifugation force may need to be reduced below 350 × g (steps 9 and 13), at the cost of a lower recovery.

Input size is constrained: a minimum of ≈40 mg of tissue and fragments larger than ≈1.5 mm in diameter are recommended for efficient dissociation. For organoids, a diameter of at least ≈2 mm is recommended for dissociation as a single unit; smaller organoids, including those at early differentiation stages, are pooled to reach a comparable input.

As a single-nucleus workflow, snRNA-seq captures nuclear transcripts only; cytoplasmic transcripts are not recovered, and the approach is not interchangeable with scRNA-seq. The downstream steps have been optimized and validated only for the EVERCODE Nuclei Fixation kit (Parse Biosciences). Compatibility with droplet-based platforms such as 10x Genomics has not been tested; the purified suspension should in principle be compatible after resuspension in the appropriate buffer, but users adopting a different platform should verify nuclei concentration and integrity in that buffer before proceeding.

Troubleshooting

Problem 1

Low nuclei concentration (related to steps 5 and 13).

If the tissue input is too small, the C Tube may not dissociate it effectively, and a single run of the program may leave macroscopic fragments undissociated, reducing nuclei yield.

Potential solution

• •Ensure the tissue is at least ≈1.5 mm in diameter; if it is smaller, increase the amount of input tissue. For organoids, the diameter should be at least 2 mm.
• •After the program, inspect the suspension — it should be turbid and homogeneous. If macroscopic fragments remain, run the 4C_nuclei program a second time immediately.

Problem 2

High proportion of debris in the suspension (related to steps 7–11).

Depending on tissue type and complexity, cellular debris can be difficult to remove completely and may persist after filtration.

Potential solution

• •After dissociation, incubate the suspension in NL Buffer on ice for an additional 5–10 min before filtration to let debris sediment.
• •After magnetic isolation, pass the column flow-through through an additional 30 μm SmartStrainer.

Note: Nuclei typically exceed 15 μm in diameter. Do not use a strainer with a pore size smaller than 20 μm, as this may cause significant nuclei loss.

Problem 3

Presence of damaged nuclei (related to step 1 and expected outcomes).

Improper collection or preservation can cause cell death and degradation of nuclear components by endogenous RNases. Damaged nuclei reduce library quality and sequencing depth.

Potential solution

• •Minimize the thaw time for snap-frozen tissue and the collection-to-processing time for live tissue, to limit exposure to endogenous RNases.
• •Add a filtration step through a smaller strainer to remove the most severely damaged nuclei.
• •If a high proportion of damaged nuclei persists, stain with a universal nuclear dye (DAPI or Hoechst) and enrich for intact nuclei by fluorescence-activated cell sorting (FACS) before fixation.

Problem 4

Presence of nuclei aggregates (related to steps 10, 14, and 17).

Free DNA and other components released from lysed cells can cause nuclei to adhere to one another, forming aggregates that reduce single-nucleus barcoding efficiency and library quality.⁴

Potential solution

• •DNase I is the key component preventing aggregation, as it digests free DNA released during dissociation. If aggregates appear, increase the DNase I concentration in the NL and NS Buffers.
• •Increase the BSA concentration to ≈1% (10 mg/mL) in the NL Buffer and up to 5% in the NS Buffer to limit non-specific interactions between nuclei.
• •Pass the suspension through a 30 μm SmartStrainer immediately before the next step to break up loose aggregates.

Problem 5

Low RNA integrity in the final library (related to buffer preparation and step 1).

RNase contamination is the main cause of degraded RNA in snRNA-seq. Even brief exposure can lower RNA integrity and reduce library complexity.⁴

Potential solution

• •Add RNase Inhibitor to all buffers immediately before use and keep everything on ice.
• •Decontaminate benches and pipettes with an RNase-removal reagent, and use certified RNase-free tips and tubes.
• •Minimize processing time and avoid repeated warming of the suspension.
• •If degradation persists, consider adding a ribonucleoside-vanadyl complex (RVC) as an additional RNase block.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anthony Flamier (anthony.flamier@umontreal.ca).

Technical contact

Technical questions on executing this protocol should be directed to the technical contact, Marion Guillon (marion.guillon@umontreal.ca).

Materials availability

This study did not generate new unique reagents. All reagents and materials used in this protocol are listed in the key resources table.

Data and code availability

This protocol does not include datasets or original code. Data supporting the associated study are reported in Guillon et al.¹ Any additional information is available from the lead contact upon request.

Acknowledgments

This work was funded by the Canada Brain Research Fund (CBRF), a partnership between Health Canada and the Brain Canada Foundation (Future Leaders Program); the Azrieli Foundation; the Canadian Institutes of Health Research; the Canadian Stem Cell Network Jump Start ECR Program; the CHU Sainte-Justine Foundation; and the Fonds de Recherche du Québec–Santé (FRQS). Additional support was provided by the Fonds UdeM pour le partenariat CHU Sainte-Justine–Institut Imagine en épilepsie de l’enfant. We thank Dr. Beauséjour’s laboratory for generously sharing equipment. The figures were created with BioRender.

Author contributions

Conceptualization, M.G. and A.F.; methodology and investigation, M.G. and M.B.; resources, L.L. and A.F.; writing – original draft, M.G.; writing – review and editing, all authors; supervision and funding acquisition, A.F.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Claude (Anthropic) in order to improve the readability, language, and structure of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.