TITLE: Fluorescence-activated nuclei sorting (FANS) for single-cell Whole Genome Sequencing (scWGS)
AUTHORS: Ester Kalef-Ezra, Lucia Friscioni, Dominic Horner, Caoimhe Morley, George Morrow, Yanping Guo, Christos Proukakis

[DESCRIPTION]
This protocol describes the isolation steps of nuclei from human post-mortem brain samples, immunofluorescence, and nuclei sorting (FANS) for low coverage (<1x) single-cell Whole Genome Sequencing (scWGS) to detect mega-base somatic Copy Number Variations (CNVs). 

We have used this protocol to isolate nuclei from the frontal cortex, cingulate cortex, and substantia nigra tissues. However, it can be adapted for nuclei from different body areas, cell culture materials, and/or samples from other species.

[BEFORE_START]
In both cases before use: Clean Human Tissue handling hood with 0.2 Molarity (M)  NaOH, 10% Presept, 70% EtOH, and dH2O.

[GUIDELINES]
Unless otherwise indicated, all the reagents must be kept at 4ºC, and perform the steps in the protocol at 4ºC. To preserve nuclear integrity, all the solutions are supplemented with complete cOmplete EDTA-free Protease Inhibitor Cocktail.

[STEPS]
SECTION: Nuclei isolation from human post-mortem brain tissue
1. In both cases prior use: Clean Human Tissue handling hood with 0.2 Molarity (M)  NaOH, 10% Presept, 70% EtOH and dH2O.
SECTION: Nuclei isolation from human post-mortem brain tissue
2. Transfer all materials needed and handle human brain samples carefully in ahuman handling hood.
 

SECTION: Nuclei isolation from human post-mortem brain tissue
4. Prepare 5% BSA in PBS (1x) and store at 4 °C.

SECTION: Nuclei isolation from human post-mortem brain tissue
5. Prepare PIC (1x complete EDTA-free Protease Inhibitor Cocktail) and store in the freezer or remove an aliquot from the freezer and place it on ice.

SECTION: Nuclei isolation from human post-mortem brain tissue
6. Use the Invent Biotech BN-020 kit according to manufacturer instructions with minor modifications as follows:

SECTION: Nuclei isolation from human post-mortem brain tissue
6.1. Cut small brain pieces of tissue of interest in pre-weighed tubes and weigh the tissue on a scale aiming for 10 mg - 30 mg of each tissue/donor depending on the downstream needs. 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.2. Add 200 µL of cold buffer A in each tube containing a tissue and place it on ice. 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.4. Incubate the tube on ice  for  5 min and carefully transfer homogenate to a filter (column) in the collection tube (avoid larger debris that sink to the bottom of the tube). 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.5. Incubate the tube with the cap open at -20 °C  for   7 min.

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.6. Cap the filter and immediately centrifuge at 13000 x g, 30 s.

SECTION: Nuclei isolation from human post-mortem brain tissue
6.7. Discard the filter (column) and resuspend the pellet by pipetting up and down gently ,10-20 times. 

 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.8. Centrifuge at 600 x g, 5 min. 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.9. Pour out the supernatant and resuspend the pellet in 200 µL PBS with 5% BSA that will be overlaid on top of buffer B in the next step.

Add 1 mLcold buffer B to a 1.5 ml eppendorf tube. 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.10. Carefully overlay the 200 µL nuclear suspension on top of buffer B by slowly expelling the nuclear suspension against the wall of the tube. 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.11. Centrifuge the tube at 1000 x g, 10 min. After centrifugation, cellular debris, oil, and myelin will stay on the top (white-milky layer). The purified nuclei are found in the pellet.

 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.12. Carefully remove the milky layer by withdrawing it into a 1 ml pipette tip and discard the rest of the supernatant. 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.13. Pour out the remaining buffer B, leaving 50 µL in the bottom of the ultracentrifuge tube (as it contains the nuclear fraction). 

SECTION: Nuclei isolation from human post-mortem brain tissue
6.14. Resuspend the pellet in 50 µL - 200 µLPBS containing Blocking Buffer. 

 

SECTION: Nuclei immunostaining
7. 
 

SECTION: Nuclei immunostaining
8. Incubate nuclei in blocking buffer for 30 min at 4 °C in a rotating wheel or in falcon tubes in a tube roller.

SECTION: Nuclei immunostaining
9. Add 1ry antibodies or conjugated antibodies directly to the blocking buffer.
SECTION: Nuclei immunostaining
10. Incubate the samples for at least 30 min  at  4 °Cwith gentle shaking.

 

SECTION: Nuclei immunostaining
11. Pellet nuclei at  800 x g, 10 min, 4 °C and carefully discard supernatant.

SECTION: Nuclei immunostaining
12. Resuspended in pre-chilled blocking buffer for a gentle wash.
SECTION: Nuclei immunostaining
13. Re-pellet nuclei at  800 x g, 10 min, 4 °C and discard supernatant.

SECTION: Nuclei immunostaining
14. Add 2ry antibodies (e.g., AF488 1:500) and DAPI (0.1 µg/ml) in blocking buffer and incubate for at least 30 min at 4 °C with gentle shaking.

 

SECTION: Nuclei immunostaining
15. Wash nuclei again with blocking buffer.
SECTION: Nuclei immunostaining
16. Pellet nuclei at 800 x g, 10 min, 4 °C and carefully discard the supernatant. Resuspend nuclei in sorting buffer and proceed to filtering using Flowmi Cell Strainers in clean 1.5 mL eppendorf tubes.

 

SECTION: Nuclei sorting (FANS)
17. Prepare 96-well collection that are compatible with your lab PCR machine by placing 5 µLof TE Buffer in each well and seal very carefully using VersiCap Mat strips.

 

SECTION: Nuclei sorting (FANS)
18. Transfer nuclei and collection plates on an ice-bucket to the sorting facility. 

 

SECTION: Nuclei sorting (FANS)
19. Acquire small amounts of nuclei from all samples and perform a batch analysis to assess the sample quality and identify the targeted populations for sorting.

 

 

 

 

SECTION: Nuclei sorting (FANS)
20. Centrifuge the collection plates for 10 s at low speed to ensure reagent at bottom. 

SECTION: Nuclei sorting (FANS)
21. Sort the single-nuclei of interest into the centrifuged 96-well collection plates.

 

SECTION: Nuclei sorting (FANS)
22. Cap the collection plates carefully to ensure all sorted cells are merged in the reagent and not attached to the side of tube. 
SECTION: Nuclei sorting (FANS)
23. Centrifuge the collection plates and place them on ice for transportation to the laboratory.

 

SECTION: Single-nucleus Whole Genome Amplification and library preparation
24. Thaw collection plates on ice. 

SECTION: Single-nucleus Whole Genome Amplification and library preparation
25. Centrifuge the collection plates briefly prior use.
SECTION: Single-nucleus Whole Genome Amplification and library preparation
26. Perform single-nucleus Whole Genome Amplification according to Takara’s guidelines. 

 

SECTION: Single-nucleus Whole Genome Amplification and library preparation
27. Prepare compatible libraries for sequencing, such as Agilent SureSelect XT HS2, Illumina DNA PCR-Free prep or other DNA library preparation kit.
SECTION: Single-nucleus Whole Genome Amplification and library preparation
28. Perform library pooling and sequence the libraries.
SECTION: Nuclei isolation from human post-mortem brain tissue
2. Before starting:

Clean Dounce tissue grinder (prior to use, between samples, and at the end of the experiment) with 0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH and dH2O, and let them dry.

SECTION: Nuclei isolation from human post-mortem brain tissue
3. UV sterilize and clean PCR hood (to be used for buffer preparation) with 10% Presept, 70% EtOH and dH2O.
SECTION: Nuclei isolation from human post-mortem brain tissue
4. Pre-cool centrifuge at 4 °C.

SECTION: Nuclei isolation from human post-mortem brain tissue
5. Prepare the NIM (Nuclear Isolation Media), ODN (Optiprep Diluent for Nuclei) buffers, 25% and 29% iodixanol solutions in a PCR hood. Keep on ice until use:

NIM (Nuclear Isolation Media):
 
 
ODN (Optiprep Diluent for Nuclei):
 

25% iodixanol solution: NIM:ODGM:ODN 6:5:1.
29% iodixanol solution: ODGM: ODN 29:31.
SECTION: Nuclei isolation from human post-mortem brain tissue
6. Transfer the buffers and Dounce tissue grinder in the human handling hood.
SECTION: Nuclei isolation from human post-mortem brain tissue
7. Transfer 20 mg -  50 mg frozen brain tissue to a microcentrifuge tube containing 500 µL of NIM using a pair of forceps.

SECTION: Nuclei isolation from human post-mortem brain tissue
8. Optional step: 
Mincing the tissue in small fragments over a glass plate on ice using a scalpel could significantly decrease the time needed for homogenization and facilitate the trituration with the pipette tips.

SECTION: Nuclei isolation from human post-mortem brain tissue
9. Gently triturate the tissue with a cut pipette tip (1ml tip). Repeat several times with pipette tips with different-cut pipette tips (estimate 3-4 different cut sizes). The tissue is perfectly dissociated once it can pass through an uncut pipette tip.

SECTION: Nuclei isolation from human post-mortem brain tissue
10. Measure the total volume using 1 ml pipette. 
SECTION: Nuclei isolation from human post-mortem brain tissue
11. Add Triton X-100 solution to the sample to achieve final concentration 0.1% (stock solution 10%).
SECTION: Nuclei isolation from human post-mortem brain tissue
12. Homogenize using 2 ml Dounce tissue grinders:

Grinder A (loose, large clearance pestles): 2-3 strokes.
Grinder B (tight, small clearance pAt this point, the sample solution should appear homogeneous, butestles): 8-12 strokes.

 

SECTION: Nuclei isolation from human post-mortem brain tissue
13. Transfer the homogenate to a fresh 1.5 mL microcentrifuge tube.
SECTION: Nuclei isolation from human post-mortem brain tissue
14. Centrifuge at 1000 x g, 8 min, 4 °C. Discard supernatant.

SECTION: Nuclei isolation from human post-mortem brain tissue
15. Resuspend the pellet gently in 700 µL 25% Iodixanol Solution.

SECTION: Nuclei isolation from human post-mortem brain tissue
16. Layer the sample (avoiding mixing the phases) onto 700 µL of 29% iodixanol solution. Use 1 mL syringe without a needle to slowly load and suspend the sample on top.

 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
17. Spin at  10300 x g, 20 min, 4 °C. Remove the supernatant, leaving 50 µL in the bottom of the ultracentrifuge tube (as it contains the nuclear fraction).

SECTION: Nuclei immunostaining
21. Pellet nuclei at  800 x g, 10 min, 4 °C and carefully discard supernatant.
Then, resuspended in pre-chilled Blocking Buffer for a gentle wash.
 

SECTION: Nuclei immunostaining
18. Incubate nuclei in Blocking Buffer for 30 min at 4 °C in a rotating wheel or falcon tubes in a tube roller.

SECTION: Nuclei immunostaining
19. Add 1ry antibodies or conjugated antibodies directly to the Blocking Buffer.
SECTION: Nuclei immunostaining
20. Incubate the samples for at least 30 min  at  4 °Cwith gentle shaking.

 

SECTION: Nuclei immunostaining
22. Re-pellet nuclei at  800 x g, 10 min, 4 °C and discard supernatant.

SECTION: Nuclei immunostaining
23. Add 2ry antibodies (e.g., AF488 1:500) and DAPI (0.1 µg/ml) in blocking buffer and incubate for at least 30 min at 4 °C with gentle shaking.

 

SECTION: Nuclei immunostaining
24. Rewash nuclei with Blocking Buffer.
SECTION: Nuclei immunostaining
25. Pellet nuclei at and carefully discard the supernatant. Resuspend nuclei in Sorting Buffer and filter the nuclei suspension using Flowmi Cell Strainers in clean 1.5 mL Eppendorf tubes.

 

SECTION: Nuclei sorting (FANS)
26. Prepare 96-well collection plates (compatible with your lab PCR machine) by placing 5 µLof TE Buffer in each well and seal them very carefully using VersiCap Mat strips or plate sealers,.

 

SECTION: Nuclei sorting (FANS)
27. Transfer nuclei and collection plates on an ice-bucket to the sorting facility. 
We use the
 

SECTION: Nuclei sorting (FANS)
28. Acquire small amounts of nuclei from all samples and perform a batch analysis to assess the sample quality and identify the targeted populations for sorting.

 

 

 

 

SECTION: Nuclei sorting (FANS)
29. Centrifuge the collection plates for 10 s at low speed to ensure reagent at bottom. 

SECTION: Nuclei sorting (FANS)
30. Sort the single-nuclei of interest into the centrifuged 96-well collection plates.

 

SECTION: Nuclei sorting (FANS)
31. Cap the collection plates carefully to ensure all sorted cells are merged in the reagent and not attached to the side of the tube. 
SECTION: Nuclei sorting (FANS)
32. Centrifuge the collection plates and place them on ice for transportation to the laboratory.

 

SECTION: Single-nucleus Whole Genome Amplification and Library Preparation
33. Thaw collection plates on ice. 

SECTION: Single-nucleus Whole Genome Amplification and Library Preparation
34. Centrifuge the collection plates briefly before use.
SECTION: Single-nucleus Whole Genome Amplification and Library Preparation
35. Perform single-nucleus Whole Genome Amplification according to Takara’s guidelines and purify the amplicons in an AMPure XP beads/sample ratio 1:1. 

 

SECTION: Single-nucleus Whole Genome Amplification and Library Preparation
36. Prepare compatible libraries for sequencing using Agilent SureSelect XT HS2, Illumina DNA PCR-Free, or another DNA library preparation kit.
SECTION: Single-nucleus Whole Genome Amplification and Library Preparation
37. Perform library pooling and sequence the libraries.
SECTION: Nuclei isolation from human post-mortem brain tissue
6.3. Homogenize the tissue using the pestle provided by grinding gently with twisting force 50-60 times. Then,  add 500 µL cold buffer A to the tube and continue to grind 20-30 times. 

SECTION: Nuclei isolation from human post-mortem brain tissue
3. Clean pestles before, between, and after use with0.2 Molarity (M) NAOH, 10% Presept and dH2O and let them air-dry before use.