TITLE: Village Nuclei Isolation With Myelin Removal
AUTHORS: Steve McCarroll, Emi Ling, Melissa Goldman, Nora Reed

[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.2srged6 and dx.doi.org/10.17504/protocols.io.bq64mzgw

[STEPS]
SECTION: Before Starting
1. Gather Supplies
Razor Blades
Glass slides
Syringes with needles (3 mL syringe with 26 1⁄2 gauge needle)
Myelin removal beads (cat # 130-096-731) https://www.miltenyibiotec.com/US-en/products/myelin-removal-beads-ii-human-mouse-rat.html#gref 
Eppendorf tubes (1.5 mL and 5 mL)
Eppendorf or Rainin pipette tips
Dry ice
20 µM filter
6 well tissue culture plate
Magnetic Bead Separation Rack for 1.5 mL tubes
RNAse free water
PBS
BSA
Lucigen RNAse inhibitor (i)
SECTION: Before Starting
1.1. Prep
Turn on and chill 1.5 mL and 50 mL centrifuges with swinging buckets to 4℃ 
Prepare all the reagents needed on ice
Clean glass slides with ethanol
Pre-chill glass slides, razors, and syringes with needles
SECTION: Before Starting
1.2. Solutions to make fresh before starting experiment
DB + 5% Kollidon 64  + 0.2 U/uL Lucigen RNAse Inhibitor (5 uL/mL)
Filtering and spinning down buffer: DB + 0.016 U/uL Lucigen RNAse inhibitor (5-12.5 uL/12.5 mLs)
     (In earlier experiments 12.5 uL of inhibitor were used, but this was later scaled down to 5 uL)
10X cell loading buffer: 1% BSA + 0.2 U/uL of i
SECTION: Tissue Homogenization
2. Section and mince tissue using a pre-chilled razor and glass slide in cryostat or on ice. Avoid including white matter as much as possible.
SECTION: Tissue Homogenization
3. Transfer minced tissue into a well of the 6-well plate containing either 1-2 mLs of extraction buffer if doing a small extraction (~600 total mg of minced tissue) or 3+ mLs of extraction buffer if doing a larger extraction (~1000 total mg of minced tissue). Mix ~20 times with 1000µL pipette.
SECTION: Tissue Homogenization
4. Incubate sample on ice for 10 minutes, mixing ~20 times with the pipette about every 2 minutes.
SECTION: Tissue Homogenization
5. Syringe the sample twice.
SECTION: Tissue Homogenization
5.1. If doing a small extraction (1-2 mLs), syringe into the same well of a 6-well tissue culture plate.
SECTION: Tissue Homogenization
5.2. If doing a larger extraction (3+ mLs), transfer the syringed tissue+buffer into a new well of a 6-well tissue culture plate each time you pass the sample through a syringe.
SECTION: Tissue Homogenization
6. Filter through a 20 uM filter, flushing with DB + i (flush the filter with 12.5 mLs DB + i for every mL of extraction buffer used).
SECTION: Tissue Homogenization
6.1. Split the volume into multiple 50 mL tubes as needed so that each tube has 12.5 mL of sample. (For a 1 mL extraction, 12.5 mLs will go into 1 tube; for a 4 mLs extraction, a total volume of 50 mLs will go into 4 tubes with 12.5 mLs per tube.)
SECTION: Tissue Homogenization
7. Spin down for 10 minutes at 4ºC - 500 x g for human/primate tissue or 600 x g for mouse.
SECTION: Tissue Homogenization
8. Remove and discard supernatants.
SECTION: Tissue Homogenization
9. Combine and resuspend the pellets in DB + i. (Use 10 mLs DB + i for a large ~1000 mg extraction.)
SECTION: Tissue Homogenization
10. Count a dilution of the nuclei.
SECTION: Myelin Removal
11. For myelin removal, dilute nuclei to 1000 n/µL with 10X cell loading buffer (1% BSA in PBS + 0.2U/uL i).
SECTION: Myelin Removal
12. Add myelin removal beads (10 uL of beads per mL of nuclei at 1000 N/uL).
SECTION: Myelin Removal
13. Mix and aliquot nuclei into 1.5 mL tubes.
SECTION: Myelin Removal
14. Incubate nuclei with rotation at 4ºC for 15 minutes.
SECTION: Myelin Removal
15. Briefly spin down the tubes to get liquid off the caps, then place on a magnet at 4ºC for 30 minutes.
SECTION: Myelin Removal
16. Transfer nuclei to new tubes, pulling up from the bottom and avoiding the myelin pellet on the side of the tube.
SECTION: Myelin Removal
17. Spin down the nuclei at 400 x g at 4ºC for 5 minutes.
SECTION: Myelin Removal
18. Remove and discard the supernatant.
SECTION: Myelin Removal
19. Resuspend the pellet in 10X cell loading buffer (1% BSA + 0.2 U/uL i).
SECTION: Myelin Removal
20. Count a dilution of the nuclei.