Jul 30, 2024
Human Fixed Neuronal Nucleus Isolation for Single-Nucleus Transcriptomic Profiling (10x Genomics)
- 1University of Texas Southwestern Medical Center;
- 2University of Texas at Dallas
Protocol Citation: Satoshi Ishishita, Katherin Gabriel, Seph Palomino, Allan-Hermann Pool 2024. Human Fixed Neuronal Nucleus Isolation for Single-Nucleus Transcriptomic Profiling (10x Genomics). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r3w4g1b/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: July 16, 2024
Last Modified: July 30, 2024
Protocol Integer ID: 103677
Keywords: single-nucleus RNA-seq, neuronal nucleus suspension, human tissue, 10x Genomics, spinal cord
Funders Acknowledgements:
UTSW Endowed Scholars Funds
Abstract
Protocol for generating suspensions of fixed neuronal nuclei (NeuN+) from human central nervous system tissue for single-nucleus transcriptomics.
Equipment and Reagents
Equipment and Reagents
Equipment
- Kimble Dounce Kontes tissue-grinder set (DWK 885300-0000)
- 50 ml Oakridge tubes (#0556214D) / can replace with 50 mL Falcon tube
- 15 mL Falcon tubes (Fisher #352097)
- 50 mL Falcon tubes (Fisher #352070)
- 1.5mL LoBind Eppendorf Tubes
- 70-micron Corning Cell Strainer (#431751)
- Fire polished glass Pasteur pipettes (VWR #14672-380, polished in an open gas flame down to ~600 micron, 300 micron and 150 micron tip opening sizes) // can replace with regular micropipetting.
Reagents
- Roche Protector RNase Inhibitor (Millipore Sigma RNAINH-RO)
- BioLegend anti-NeuN antibody (#608452, for labeling neuronal nuclei)
- Alexa Fluor 647 Microscale Protein Labeling Kit (ThermoFisher #A30009)
- 1 mg/ml 7-AAD (nuclear labeling)
- 1M DTT (dithiothreitol, prepare fresh every couple of months and store at -20°C)
- Ultrapure RNA-se free/ DNA-se free water
Protocol outline
Figure 1: Protocol outline with spinal cord as sample central nervous system tissue.
Solutions
Solutions
NMDG-Hepes-ACSF
- NMDG (93 mM)
- KCl (2.5 mM)
- NaH2PO4 (1.2 mM)
- NaHCO3 (30 mM)
- HEPES (20 mM)
- Glucose (25 mM)
Bring pH to between 7.3 - 7.4 with 10N HCl and filter sterilize (good for 2 weeks at 4°C).
On the morning of tissue preparation add the following components (final concentration):
- Na-Ascorbate (5 mM)
- Thiourea (2 mM)
- Na-pyruvate (3 mM)
- MgSO4 (10 mM, prepare 2M stock that is good for 6-months at 4°C)
- CaCl2 (1 mM, prepare 2M stock that is good for 6-months at 4°C)
- Kynurenic acid Na-salt (1 mM)
Nuclear Buffer
- Sucrose (320 mM)
- Tris-HCl (pH=7.4) (10 mM)
- MgCl2 (3 mM)
- NaCl (10 mM)
- BSA (RNAse free) (0.50%)
- Kollidon VA64 (1 %)
- Ultrapure water, fill to 50 mL and 0.22 micron filter sterilize.
Morning of run:
- DTT (dithiothreitol, 1 mM)
- Roche Protector RNAse Inhibitor (0.1 U/uL)
Lysis Buffer
- Nuclear buffer
- Triton-X100 (0.1%)
1M Sucrose Cushion
Sucrose (1 M)
Tris-HCl (pH=7.4) (10 mM)
MgCl2 (3 mM)
NaCl (10 mM)
BSA (nuclease free) (0.50%)
Kollidon VA64 (1%)
Water (molecular biology grade) Fill to 50 mL
Do NOT Filter sterilize!
Protocol
Protocol
1. Prepare solutions and equipment
- Prepare 50 mL of NMDG-HEPES-ACSF from pre-prepared stock by adding (Na-Ascorbate, Thiourea, Na-pyruvate, MgSO4, CaCl2 and Kynurenic acid Na-salt) and place on ice.
- Prepare Nuclear Buffer (add DTT and RNA-se inhibitor to preprepared solution) and place on ice.
- Prepare Lysis Buffer from Nuclear Buffer (add Triton-X100 to 0.1% of final volume) and pipette 0.75 mL into a Kontes tissue grinder.
- Prepare 1M sucrose cushion (add DTT and RNA-se inhibitor to preprepared solution) and place on ice.
- Pre-cool centrifuge to 4°C.
- Place 100 mm dissection dish into a 150 mm dish with dry ice.
2. Dissect out tissue
Place snap frozen brain tissue into 100 mm tissue culture dissection dish on a layer of dry ice in a larger 150 mm dish. Microdissect out desired tissue parts and cut into small 1.5 mm3 cubicles. Drop the latter into ice-cold NMDG-Hepes-ACSF in 1.5 mL collection tubes on ice.
3. Generate nuclear suspension
- Transfer tissue pieces into the Lysis Buffer in the Kontes tissue grinder.
- Apply 5 strokes with the loose pestle followed by 15 strokes with the tight pestle.
- Place a 70-micron cell strainer on a 50 mL Falcon tube and pre-wet with 500 μL of Nuclear Buffer.
- Add 250 μL of Nuclear Buffer to the tissue grinder.
- Mix nuclear suspension in tissue grinder twice with a 600-micron fire polished glass capillary and transfer through the cell strainer.
- Wash tissue grinder with 750 μL Nuclear Buffer and transfer again through the cell strainer.
- Wash cell strainer with final 750 μL Nuclear Buffer.
4. Spin nuclei down and resuspend in fresh Nuclear Buffer
- Spin nuclei down for 5 min at 500g at 4°C in a spin-out rotor.
- Remove supernatant and resuspend in fresh 3 mL Nuclear Buffer.
5. Purify nuclei with a sucrose cushion centrifugation
- Transfer 12 mL of Sucrose Cushion into a 50 mL Oakridge tube.
- Gently layer the nuclear suspension from the previous step on the sucrose cushion (avoid mixing of the layers).
- Centrifuge the tubes at 3200g at 4°C for 20 minutes in a spin-out rotor.
6. Fix nuclei
- After centrifugation, pour out the supernatant by decanting in one smooth motion and drying out the neck of the Oakridge tube with a Kimwipe.
- Resuspend nuclei with 1mL of Fixation buffer from the 10X fixation of cells & nuclei for chromium fixed RNA profiling (CG000478) and transfer resuspension to a 15mL Falcon tube and incubate for 18hr at 4°C.
7. Stain nuclei for FACS sorting
- Centrifuge tube at 500g for 5min at 4°C to wash off the fix.
- Resuspend in 300 μL of Nuclear Buffer.
- Add 3 μL of anti-NeuN-Alexa-647 Ab (conjugate Alexa-647 to Biolegend anti-NeuN antiobody prior to use) and 3 μL of 7-AAD to the nuclear suspension.
- Incubate the nuclear suspension at 4°C (not on ice!; keep dark as 7-AAD is light sensitive).
- Bring nuclear suspension volume to 3 mL with Nuclear Buffer.
- Spin nuclei down at 500g for 5 min at 4°C in a spin-out rotor.
- Remove most of supernatant and resuspend nuclei in 3 mL of Nuclear Buffer.
- Place suspension at 4°C for 5 min.
- Spin nuclei down at 500g for 5 min at 4°C.
- Remove supernatant and resuspend nuclei in 0.5 mL of Nuclear Buffer.
- Gently triturate with 150 micron glass Pasteur pipette to declump any nuclei.
- Add 1 mL of Nuclear Buffer and take to FACS sorting.
8. FACS sorting
- Sort at low pressures (4 or 5 PSI on BD sorters).
- Select for 7-AAD and Alexa 647 double-positive nuclei (note - perform compensation with single-stains of dyes before the actual profiling run to get clear separation between double-positive neuronal nuclei and single-positive non-neuronal nuclei).
Figure 2: Sorting gates for isolating neuronal nuclei (double positive for 7-AAD and Alexa-647)
9. Store or prepare nuclei for profiling
- Post FACS sorting, spin collected nuclei at 850g for 5 min at 4°C and resuspend the nuclei with 1 mL of quenching buffer and follow the instructions of the 10X fixed nucleus profiling kit to freeze the samples long term or short-term depending on the timeline for sequencing steps.
- Proceed to 10x Genomics or other profiling.