Village Nuclei Isolation With Optiprep

Gather Supplies

• Scalpels

• Glass slides

• 14 mL Dounce

• 20 µm vacuum filter

• Eppendorf tubes (1.5 mL and 5 mL)

• Eppendorf or Rainin pipette tips

• Dry ice

• Metal plate

• OCT (Optimal cutting temperature compound)

• RNAse free water

• Cell counting supplies (LUNA-FL) Other Reagents:

• PBS

• BSA

• RNAse inhibitor (i)

• Nuclei EZ lysis buffer (NUC201-1KT)

• OptiPrep Density Gradient Medium (60% Iodixanol - G60) (ab286850) Stock solutions:

• 500 mM tricine : 8.96 g of tricine in 100 mL water

• 1 M KCl : 7.45 g of KCl in 100 mL water

• 1 M MgCl2x6H2O : 20.3 g of MgCl2x6H2O in 100 mL water

Optiprep Buffers (can be made in advance and kept on hand)

A	B	C	D
GD		GH
Component	Volume	Component	Volume
500 mM Tricine	24 mL	Sucrose	8.5 g
1M KCl	15 mL	Water	50 mL
1M MgCl2 x 6H2O	3 mL	500 mM Tricine	4 mL
Water	50 mL	KCl	2.5 mL
1M KOH	Adjust to pH 7.8	1M MgCl2x6H2O	0.5 mL
	Bring volume up to 100 mL with water (keep at room temp)	1M KOH	Adjust to pH 7.8
			Bring volume up to 100 mL with water (keep at 4°C)

Turn on and chill centrifuge to 4℃* Prepare all the reagents needed on ice

• Clean glass slides with ethanol
• Gather the dounce, pestles, and scalpels on ice

Solutions to make fresh before starting experiment

Nuclei EZ lysis buffer

• Pour 12 mL of buffer into the dounce and reserve 1 mL for wetting down the sides of the dounce PBSA + i (1% BSA and 1 U/µL RNase inhibitor in 1X PBS)

• For powder BSA, add 0.01 g of BSA per 1 mL of 1X PBS G30: (Mix the G60 and GD together thoroughly before adding the GH)

A	B	C	D	E	F
G30		PBSA + i		Nuclei EZ Lysis Buffer
Component	Volume	Component	Volume	Component	Volume
G60 (OptiPrep)	6.0 mL	PBS (pH 7.4) (1X)	1 mL of 10X PBS	EZ Lysis Buffer	12,975 µL
GD	1.2 mL	BSA (100X)	1 mL of 10% BSA	RNAse Inhibitor	325 µL
GH	4.8 mL	RNase Inhibitor (200X)	50 µL
		RNase free water	7,950 µL

On a glass slide on dry ice , shave off pieces of tissue with a scalpel until you have 50±3 mg of tissue

For managing tissue, use OCT to adhere tissue to glass slide

Place tissue pieces on a boat on dry ice. Hold the tissue on dry ice until all tissue has been cut

Transfer all tissue to dounce filled with Nuclei EZ Lysis Buffer

Use 1mL of the reserved Nuclei EZ Lysis Buffer to wet down the side of the dounce if tissue is stuck on the side of the dounce

Gently dounce tissue on ice 20 times with pestle “A” then 20 times with pestle “B” until the tissue is homogenized

Incubate on ice for 10 minutes

Wet a 20 µm vacuum filter with 1 mL of PBSA + i

Vacuum filter the homogenized tissue

If the filter clogs, remove any unfiltered volume with a pipette, replace the filter, and continue filtering

Transfer the homogenized tissue to (3) 5 mL tubes

Centrifuge the samples at 4℃ for 5 min (500 x g)

Remove and discard the supernatant (be careful not to disturb cell pellets) and resuspend each pellet in 900 µL G30

Alternatively, split pellet into (9) 1.5 mL tubes (total), resuspend each pellet in 300 µL of G30, then underlay each sample with 1 mL of G30

Gently underlay each 5mL tube with 3 mL G30

Place the pipette tip filled with G30 at the bottom of the 5mL tube. Slowly dispense G30 underneath the cell suspension. A clear separation will form between homogenate and G30 (cloudy on top, clear on the bottom). Do not disturb the separation between the layers

Centrifuge the samples at 4℃ for 10 min (4,255 x g)

For capturing smaller cell types, spin at 8,000 x g for 20 minutes

Remove and discard the supernatant

Remove the top (very viscous) layer from the tube first- this layer contains fats and must be removed before removing the rest of the supernatant

Resuspend the pellets in 1 mL PBSA + i

Centrifuge the samples at 4℃ for 5 min (500 x g) (single wash)

Remove the supernatant and resuspend the pellets in 200 - 1,000 µL of PBSA + i (depending on the size of the pellet)

Count nuclei using LUNA-FL counter

Optional if you have excess nuclei, or if you need to pause before continuing.

Centrifuge the nuclei at 4℃ for 5 min (500 x g)

Remove the supernatant and resuspend the nuclei in CryoStor (CryoStor® CS10), mix by pipetting

≤ 1k nuclei / µL of CryoStor

Transfer the nuclei to cryotubes and store them in a Mr Frosty in a -80℃ freezer until frozen (1 day)

For long term storage, transfer cryotubes to regular storage in a -80℃ freezer

Thaw the CryoStored nuclei

Centrifuge the nuclei at 4°C for 10 min (1200 x g)

Remove the supernatant and resuspend in 1mL PBSA + i

Centrifuge the nuclei at 4°C for 5 min (500 x g) (single wash)

Remove the supernatant and resuspend the pelleted nuclei in PBSA + i

Count the nuclei before proceeding to next steps