Human Fixed Neuronal Nucleus Isolation for Single-Nucleus Transcriptomic Profiling (10x Genomics)

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

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!

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 X 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).

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.