TITLE: Human Fixed Neuronal Nucleus Isolation for Single-Nucleus Transcriptomic Profiling (10x Genomics)
AUTHORS: Satoshi Ishishita, Katherin Gabriel, Seph Palomino, Allan-Hermann Pool

[DESCRIPTION]
Protocol for generating suspensions of fixed neuronal nuclei (NeuN+) from human central nervous system tissue for single-nucleus transcriptomics.

[STEPS]
SECTION: Equipment and Reagents
1. 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.
SECTION: Equipment and Reagents
2. 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
SECTION: Solutions
4. 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)
SECTION: Solutions
5. 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)
SECTION: Solutions
6. Lysis Buffer
Nuclear buffer
Triton-X100 (0.1%) 
SECTION: Solutions
7. 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!			

SECTION: Protocol
8. 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.
SECTION: Protocol
9. 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.
SECTION: Protocol
10. 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. 
SECTION: Protocol
12. 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.
SECTION: Protocol
14. 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.
SECTION: Protocol
15. 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).


 

SECTION: Protocol
11. 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.
SECTION: Protocol
16. 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.
SECTION: Protocol
13. 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.
SECTION: Equipment and Reagents
3. Protocol outline