TITLE: Protocol for Preparing Brain Samples for MUSIC
AUTHORS: Wenxin Zhao, Sheng Zhong

[DESCRIPTION]
Here states the detailed procedure to prepare brain samples for MUSIC study.

[STEPS]
SECTION: Tissue pulverization and crosslinking
1. Cut a portion of post-mortem human brain frontal cortex sample collected from The Brain and Body Donation Program (BBDP) at Banner Sun Health Research Institute on dry ice with heavy razor blades, and collect 50 mg of the sample in a 1.5 mL LoBind tube. 
SECTION: Nuclei isolation
8. Use Chromium Nuclei Isolation kit (10X genomics, 1000494) to isolate nuclei from crosslinked cortex samples. 
SECTION: Nuclei isolation
9. Transfer 50 mg frozen tissue into pre-chilled sample dissociation tube. 
SECTION: Nuclei isolation
10. Add 400 μL of Lysis Buffer to Sample Dissociation Tube. Dissociate tissue with plastic pestle until homogeneous. 

 

SECTION: Nuclei isolation
12. Equally load the solution into two nuclei isolation column, and centrifuge the tubes at 16,000 x g for 20 sec at 4°C.
SECTION: Nuclei isolation
13. Vortex the flowthrough in the collection tube that contains nuclei for 10 sec at 3,200 rpm or max speed to resuspend nuclei. 
SECTION: Nuclei isolation
14. Centrifuge the collection tubes for 3 min at 500 x g at 4°C to pellet nuclei. Carefully discard the supernatant. 
SECTION: Tissue pulverization and crosslinking
2. Thaw the 50 mg of brain sample on ice, and chop the tissue into smaller pieces by pestle. Store the rest of the sample at -80°C.
SECTION: Tissue pulverization and crosslinking
3. Incubate the sample with 10 mL of 2 mM disuccinimidyl glutarate (DSG) in 1X PBS in a 15 mL LoBind tube at room temperature for 45 min with gentle rotation. 
SECTION: Tissue pulverization and crosslinking
4. Wash once with 10 mL of 1X PBS by centrifugation at 1,000 x g for 4 min.
SECTION: Tissue pulverization and crosslinking
5. Resuspend the sample in 15 mL of 1X PBS containing 3% formaldehyde, and incubate for 10 min with a gentle rotation.
SECTION: Tissue pulverization and crosslinking
6. Quench the crosslinking reaction by the addition of 5 mL of 1.25 M glycine followed by an incubation of 5 min with a rotation.
SECTION: Tissue pulverization and crosslinking
7. Centrifuge the sample at 1,000 x g for 4 min, and wash the sample twice with ice-cold 1X PBS containing 0.3% BSA (wt/vol).
SECTION: Nuclei isolation
11. Add 600 µL of lysis buffer into the tube, and mix 10 times by pipetting. Incubate on ice for 10 min.
SECTION: Nuclei isolation
15. Resuspend the nuclei in 500 µL of Debris Removal Buffer provided by the kit by pipetting 15 times. 

 

SECTION: Nuclei isolation
16. Centrifuge the nuclei at 700 x g for 10 min at 4°C. Carefully discard the supernatant. 
SECTION: Nuclei isolation
17. Resuspend the nuclei in 1 mL of Wash and Resuspension Buffer. 

 

SECTION: Nuclei isolation
18. Centrifuge the nuclei at 500 x g for 5 min at 4°C. Carefully discard the supernatant. 
SECTION: Nuclei isolation
19. Resuspend the nuclei again in 1 mL of Wash and Resuspension Buffer. 
SECTION: Nuclei isolation
20. Centrifuge the nuclei at 500 x g for 5 min at 4°C. Carefully discard the supernatant as much as possible. 
SECTION: Nuclei isolation
21. The nuclei are subjected to nuclei counting and the following procedures.