TITLE: Village Nuclei Isolation With Optiprep
AUTHORS: liv_spina, Tara McDonald, Nora Reed, Alyssa Lutservitz

[DESCRIPTION]
Isolation of nuclei from fresh-frozen brain tissue from sets of multiple (typically 2-20) human donors for analysis as a “cell village” (Wells et al., PMID 36796362) in which nuclei from all donors are analyzed together. Adapted from dx.doi.org/10.17504/protocols.io.bs99nh96, Luciano G Martelotto, with modifications to optimize for human brain tissue and allow the “cell village” approach.

[BEFORE_START]
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)

[STEPS]
SECTION: Before Starting
1. 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
SECTION: Before Starting
1.1. Optiprep Buffers (can be made in advance and kept on hand)
    GDGHComponentVolumeComponentVolume500 mM Tricine24 mLSucrose8.5 g1M KCl15 mLWater50 mL1M MgCl2 x 6H2O3 mL500 mM Tricine4 mLWater50 mLKCl2.5 mL1M KOHAdjust to pH 7.81M MgCl2x6H2O0.5 mLBring volume up to 100 mL with water (keep at room temp)1M KOHAdjust to pH 7.8Bring volume up to 100 mL with water (keep at 4°C)

 
 
SECTION: Before Starting
1.2. 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
SECTION: Before Starting
1.3. 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)

 
 
      G30PBSA + iNuclei EZ Lysis BufferG60 (OptiPrep)6.0 mLPBS (pH 7.4) (1X)1 mL of 10X PBSEZ Lysis Buffer12,975 µLGD1.2 mLBSA (100X) 1 mL of 10% BSARNAse Inhibitor325 µLGH4.8 mLRNase Inhibitor (200X) 50 µLTotal 12 mLRNase free water7,950 µL

 
 
SECTION: Nuclei Isolation
7. Wet a 20 µm vacuum filter with 1 mL of PBSA + i
SECTION: Tissue Homogenization
2. On a glass slide on dry ice, shave off pieces of tissue with a scalpel until you have 50±3 mg of tissue
SECTION: Tissue Homogenization
2.1. For managing tissue, use OCT to adhere tissue to glass slide
SECTION: Tissue Homogenization
4. Transfer all tissue to dounce filled with Nuclei EZ Lysis Buffer
SECTION: Tissue Homogenization
5. Gently dounce tissue on ice 20 times with pestle “A” then 20 times with pestle “B” until the tissue is homogenized
SECTION: Tissue Homogenization
6. Incubate on ice for 10 minutes
SECTION: Tissue Homogenization
4.1. 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
SECTION: Tissue Homogenization
3. Place tissue pieces on a boat on dry ice. Hold the tissue on dry ice until all tissue has been cut
SECTION: Nuclei Isolation
8. Vacuum filter the homogenized tissue
SECTION: Nuclei Isolation
9. Transfer the homogenized tissue to (3) 5 mL tubes
SECTION: Nuclei Isolation
8.1. If the filter clogs, remove any unfiltered volume with a pipette, replace the filter, and continue filtering
SECTION: Nuclei Isolation
10. Centrifuge the samples at 4℃ for 5 min (500 x g)
SECTION: Nuclei Isolation
11. Remove and discard the supernatant (be careful not to disturb cell pellets) and resuspend each pellet in 900 µL G30 
SECTION: Nuclei Isolation
11.1. 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
SECTION: Nuclei Isolation
12. 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
SECTION: Nuclei Isolation
13. Centrifuge the samples at 4℃ for 10 min (4,255 x g)
SECTION: Nuclei Isolation
13.1. For capturing smaller cell types, spin at 8,000 x g for 20 minutes
SECTION: Nuclei Isolation
14. Remove and discard the supernatant
SECTION: Nuclei Isolation
14.1. 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
SECTION: Nuclei Wash, Resuspension, and Quantification
15. Resuspend the pellets in 1 mL PBSA + i
SECTION: Nuclei Wash, Resuspension, and Quantification
16. Centrifuge the samples at 4℃ for 5 min (500 x g) (single wash)
SECTION: Nuclei Wash, Resuspension, and Quantification
17. Remove the supernatant and resuspend the pellets in 200 - 1,000 µL of PBSA + i (depending on the size of the pellet)
SECTION: Nuclei Wash, Resuspension, and Quantification
18. Count nuclei using LUNA-FL counter
SECTION: Preserving Leftover Nuclei
19. Optional if you have excess nuclei, or if you need to pause before continuing. 
SECTION: Preserving Leftover Nuclei
20. Centrifuge the nuclei at 4℃ for 5 min (500 x g)
SECTION: Preserving Leftover Nuclei
21. Remove the supernatant and resuspend the nuclei in CryoStor (CryoStor® CS10), mix by pipetting 
SECTION: Preserving Leftover Nuclei
21.1. ≤ 1k nuclei / µL of CryoStor
SECTION: Preserving Leftover Nuclei
22. Transfer the nuclei to cryotubes and store them in a Mr Frosty in a -80℃ freezer until frozen (1 day)
SECTION: Preserving Leftover Nuclei
23. For long term storage, transfer cryotubes to regular storage in a -80℃ freezer
SECTION: Thawing Cryo-Stored Nuclei
24. Thaw the CryoStored nuclei
SECTION: Thawing Cryo-Stored Nuclei
25. Centrifuge the nuclei at 4°C for 10 min (1200 x g)
SECTION: Thawing Cryo-Stored Nuclei
26. Remove the supernatant and resuspend in 1mL PBSA + i
SECTION: Thawing Cryo-Stored Nuclei
27. Centrifuge the nuclei at 4°C for 5 min (500 x g) (single wash)
SECTION: Thawing Cryo-Stored Nuclei
28. Remove the supernatant and resuspend the pelleted nuclei in PBSA + i
SECTION: Thawing Cryo-Stored Nuclei
28.1. Count the nuclei before proceeding to next steps