Human Fixed 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 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

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

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%)

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!

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.

• 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.

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

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.