TITLE: Human Fixed 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 human nuclei 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 Tubes
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) // alternatively can replace with regular pipetting
SECTION: Equipment and Reagents
2. Reagents
Roche Protector RNase Inhibitor (Millipore Sigma RNAINH-RO)
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. 1.8M Sucrose Cushion
Sucrose	                   (1.8 M)
Tris-HCl (pH=7.4)	   (10 mM)
MgCl2                         (3 mM)
NaCl                           (10 mM)
BSA (nuclease free)  (0.50%)
Kollidon VA64            (1%)

Water (ultrapure)			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.
- 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 the nuclear pellet in 100 μL of ice-cold Nuclear Buffer and mix gently with a 300 micron fire polished Pasteur pipette.
- Transfer purified nuclear suspension to a new 15 mL tube on ice.
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
14. 7. Fix nuclei and proceed with profiling
- Remove supernatant and resuspend nuclei in 1mL of Fixation buffer from the 10X fixation
of cells & nuclei for Chromium fixed RNA profiling (CG000478) and transfer resuspension to a microcentrifuge tube and incubate for 18hr at 4°C. 
- Continue with 10x fixation protocol.
SECTION: Equipment and Reagents
3. Protocol Outline

 

SECTION: Protocol
13. 6. Evaluate debris
-  Pipette 3 μL of the nuclear suspension on a glass slide and evaluate debris under a brightfield microscope
-  If there is a lot of debris, add 2ml of Nuclear Buffer to the nuclear suspension and spin nuclei down at
500g for 5 min at 4C in a spin-out-rotor.