Nuclei Isolation

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NEB (Nuclei Extraction Buffer)

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NSB (Nuclei Suspension Buffer)

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BSA (25% Bovine Serum Albumin)

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DWB (Dounce Wash Buffer)

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RNI (RNase Inhibitor, 40 U/µl)

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Acridine Orange and Propidium Iodide (AO/PI)

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Cell strainer, 40 μm

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Conical tube, sterile and nuclease-free, 5 ml

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Conical tube, sterile and nuclease-free, 15 ml

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Conical tube, sterile and nuclease-free, 50 ml

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Luer-Lok Syringe, 5 ml

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Nuclease-free water

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Pipette tips, filtered, low-retention, sterilized, 20 μl

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Pipette tips, filtered, low-retention, sterilized, 200 μl

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Pipette tips, filtered, low-retention, sterilized, 1000 μl

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Pipette tips, filtered, low-retention, sterilized, wide-orifice, 1000 μl

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Protease inhibitor mini-tablets

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Serological pipettes, 5 ml

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Serological pipettes, 10 ml

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Tube, sterile and nuclease-free, 1.5 ml

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One of the following filter sizes based on nuclei size distribution:

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ÜberStrainer 10 µm

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ÜberStrainer 15 µm

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ÜberStrainer 20 µm

Make sure that all reagents are fully thawed and well-mixed before use.

1.

Prepare the centrifuge as follows.

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Prechill the centrifuge to 4°C.

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Balance the centrifuge before loading samples.

2.

Prepare the following consumables:

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10X Protease Inhibitor Solution—Dissolve one tablet in 1 ml nuclease-free water and vortex to mix.

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Nuclei Wash Buffer (NWB)—Dilute 1 ml NSB in 5 ml nuclease-free water and vortex to mix.

3.

Combine the following volumes to prepare 4 ml NEB working solution:

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NEB (2000 μl)

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RNI (80 μl)

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10X Protease Inhibitor Solution (400 μl)

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Nuclease-free water (1520 μl)

4.

To prepare the NSB working solution, refer to the following tables for the appropriate volume of NSB for the input tissue type and mass.

The suggested mass-to-volume ratios are based on a final concentration of ~5000 nuclei/μl. Adjust resuspension or dilution volume accordingly to achieve the desired concentration for different kit sizes.

Recommended Ratios of NSB Working Solution per mg Tissue

Tissue Type	NSB Working Solution per mg Tissue (μl)
Brain	20
Liver	7
Lung	5
Heart	0.5
Diaphragm	1

If you have an unvalidated tissue type, you may need to optimize NSB working solution volume for your sample.

1000 μl NSB Working Solution Formulation Example

Component	Fresh Nuclei (μl)	Fixed Nuclei (μl)
NSB	167	167
25% BSA	40	0
RNI	20	0
Nuclease-free water	773	833
Total	1000	1000

If planning on fixing nuclei after isolation using the DSP-Methanol Fixation for Nuclei demonstrated protocol, do not add BSA or RNI to your NSB working solution. BSA and RNI are incompatible with the fixation protocol.

1.

Add 2 ml chilled DWB into three 5 ml tubes. Place the individual dounce pieces in each tube.

2.

Record the tare mass of a 1.5 ml tube. Place this tube on ice.

3.

Obtain a section of the tissue of interest, place it in the chilled 1.5 ml tube, and then do as follows.

a.

Record the mass of the tube with the section of tissue inside.

b.

Calculate the mass of the tissue.

Make sure that the mass does not exceed the maximum input for that tissue type.

4.

Add 1 ml ice-cold NEB working solution to the tissue tube.

5.

Use a wide-orifice 1000 μl pipette tip to transfer this material into the cap of the 50 ml conical tube.

6.

Cut the sample into < 2–3 mm pieces.

7.

Using a wide-orifice 1000 μl pipette tip, transfer to the dounce homogenizer.

8.

Dounce eight times with the loose pestle, and then dounce eight times with the tight pestle.

If the tissue chunks are difficult to separate or visible clumping is observed, gently rotate the pestle 180° clockwise during douncing. If there are still visible chunks of tissue left after processing with the loose pestle, consider increasing the number of strokes performed.

If the sample is fragile, consider decreasing the number of strokes performed.

9.

Place the 40 μm cell strainer on top of a 50 ml conical tube.

10.

Pass the material in the dounce homogenizer through the cell strainer.

11.

Wash the dounce homogenizer with 3 × 1 ml NEB and pass through the same cell strainer.

12.

Transfer the 4 ml total filtrate to a 15 ml conical tube.

13.

Incubate the sample on ice for 5 minutes.

If you have an unvalidated tissue type, you may need to optimize incubation time for your sample. More sensitive samples may require a shorter incubation time. More robust samples may require a longer incubation time.

14.

Centrifuge the dissociated nuclei at 500 × g for 5 minutes at 4°C (Acceleration 9, Deceleration 7).

15.

Remove and discard the supernatant.

16.

Add 1 ml NWB into the tube containing the nuclei pellet.

17.

Using a 1000 μl pipette tip, gently break and resuspend the nuclei pellet.

18.

Using a serological pipette, add 4 ml NWB on top of the 1 ml suspension and mix well.

19.

Assemble the Überstrainer on a 50 ml conical tube, ensuring a tight seal. Use the appropriate Überstrainer filter size for your sample.

20.

Attach a 5 ml syringe to the Luer-Lock adapter on the side of the strainer.

21.

Pipette 2 ml nuclei suspension into the strainer.

22.

To pass the suspension through the strainer, slowly apply negative pressure with the syringe at a rate of 1 ml per 5 seconds or slower.

Do not exceed the 3 ml line on the syringe. If close to hitting that mark, detach the syringe, reset the plunger to 0, and then reattach the syringe.

23.

Add the remainder of the nuclei suspension. Continue to filter until there is no liquid remaining on the filter but the filter still looks moist.

24.

Transfer the filtrate to a 15 ml conical tube.

25.

Centrifuge nuclei at 500 × g for 5 minutes at 4°C (Acceleration 9, Deceleration 7).

26.

Remove and discard the supernatant.

27.

Resuspend the nuclei pellet in the prepared NSB working solution.

If you plan on fixing nuclei after isolation using the DSP-Methanol Fixation for Nuclei protocol, make sure that the prepared NSB working solution does not contain BSA or RNI. These reagents are incompatible with the fixation protocol.

28.

Using a 200 μl pipette, gently pipette to resuspend the nuclei fully.

Use a minimum number of pipetting strokes.

29.

Quantify samples using your preferred method.

For accurate quantification, Illumina recommends fluorescent staining with AO/PI and an automated fluorescent cell counter. When staining with AO/PI, use the red channel (dead) count for the nuclei counting.

30.

To use the nuclei with a compatible Illumina Single Cell 3′ RNA Prep kit, follow the instructions in the applicable product documentation.

Refer to the following table for the target nuclei concentration for the respective Illumina Single Cell 3' RNA Prep kit size:

Kit Size	Optimal Nuclei Input*	Expected Capture Rate	Nuclei Loading Volume (µl)	RNase Inhibitor Volume (µl)	Target Concentration
T2	5000	≥ 2000	4	1	1250 nuclei/µl
T10	17,000	≥ 10,000	5	1	3400 nuclei/µl
T20	40,000	≥ 20,000	8	1–2	5000 nuclei/µl
T100	200,000	≥ 100,000	20	4	10,000 nuclei/µl

*Exceeding the recommended input increases the risk of a multiplet rate above 8%