Nuclei isolation

Nuclei isolation protocol for mouse placenta

Adapted from - https://www.protocols.io/view/frankenstein-protocol-for-nuclei-isolation-from-f-3fkgjkw/abstract

Blelloch Lab – Bryan Marsh

Buffers and Reagents

1. Nuclei EZ Lysis Buffer (Millipore Sigma) (chilled, 4°C)

2. Nuclei wash and resuspension buffer (prepare chilled, 4°C)

1x PBS

1.0% BSA

0.2 U/μl RNase Inhibitor

3. Nuclei wash and resuspension buffer with DAPI (prepare chilled, 4°C)

1x PBS

1.0% BSA

0.2 U/μl RNase Inhibitor

10 ug/mL DAPI

4. 10x v3 RT Buffer1 for Single Cell Gene Expression 3’ reagents (DO NOT add RT enzyme)

RT Reagent Mix: 20.0 uL

Template Switch Oligo: 3.1 uL

Reducing Agent B: 2.0 uL

H₂O: 27.1 (46.6 – 19.5) uL

Volume of 17,500 sorted nuclei: 19.5ul

After sorting add 8.3ul RT Enzyme C

Total: 80ul

Protocol

NOTE: All samples and reagents are kept on ice or at 4 °C 

1. Dissect out labyrinth (or villi) from decidual stroma under dissecting scope.

2. Mince/chop tissue into small pieces with a razor blade and forceps. Aim for volume of <1mm³ for all pieces of tissue

3. Add 2mL cold Nuclei EZ Lysis Buffer to the tissue in 7mL dounce homogenizer on ice. Homogenize the sample using a douncer (stroking ~10 with both loose and tight pestle).

4. Transfer the homogenate to a 15mL falcon tube and add 2mL of chilled Nuclei EZ Lysis Buffer, mix gently and incubate on ice for 5 min. Gently mix with a wide bore tip and repeat 1-2 times during the incubation.

5. Filter homogenate using a 35μm mesh filter and transfer to a new 15mL falcon tube. 

6. Centrifuge the nuclei at 500g for 5 minutes at 4°C and remove supernatant leaving behind ~50 uL. Gently resuspend nuclei in another 1.5 mL of EZ Lysis buffer, incubate for 5 minutes on ice.

7. Centrifuge the nuclei at 500g for 5 min at 4°C, remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~50 uL behind).

8. Add  1mL Nuclei Wash and Resuspension Buffer and incubate 5 minutes without resuspending to allow buffer interchange. After incubation, add 1 mL of Nuclei Wash and Resuspension Buffer and resuspend the nuclei.

9. Centrifuge the nuclei at 500g for 5 min at 4°C, remove supernatant leaving behind ~50 uL and gently resuspend nuclei in 1 mL Nuclei Wash and Resuspension Buffer. 

Keep in a 15mL falcon tube. Centrifuging w/ a swinging bucket rotor is important going forward (spinning in 1.5mL Eppendorf tubes w/ a tabletop minicentrifuge results in nuclei pelleted against the tube wall leading to loss of the nuclei).

10. Filter nuclei through 35-μm cell strainer into FACS tubes. Visually inspect nuclei integrity under a microscope.

11. Nuclei can be washed or filtered again (through a 35um or even a 20um) to remove debris or clumped nuclei.

12. Add 1uL of 1000x DAPI to the final suspension.

13. FACS sort on Aria II w/ a 70um nozzle. Sort for singleton nuclei w/ a nice DNA content plot (should see clear 2n, 4n, and 8n peaks).

14. Sort 17,500 nuclei into a 96-well plate preloaded with 3 wells containing 52.2uL of Nuclei Wash and Resuspension Buffer.

15. Measure the volume in these wells to ensure it is now 71.7 uL

16. Check to make sure the solution contains nuclei w/ either cell counter or hemocytometer.

17. If the volumes in the test wells are correct and consistent and nuclei are present, load RT master mix (w/o RT Enzyme C) into one well on the 96 well plate. Sort 17,500 nuclei into this well.

18. Transfer 71.7ul of the Master mix + nuclei to a 10x safe PCR tube and add 8.3uL of RT Enzyme C. Total volume is now 80uL

19. Load 10x Chip and capture as soon as possible. Excess time will lead to RNA loss from nuclei and/or degradation.

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