TITLE: Human Brain Nuclei Isolation Using Sucrose Gradient Ultracentrifugation
AUTHORS: Zhang Lab

[DESCRIPTION]
This protocol describes about isolation of human brain nuclei by sucrose gradient ultracentrifugation.

[STEPS]
SECTION: Isolation of Human Brain Nuclei
1. Take brain tissue sample out of -80 °C freezer and put on dry ice. Transfer the sample from the bag or tube to a petri dish and carefully cut a small piece off with a surgical blade, keeping it on dry ice. Replace the remaining piece in the bag and put the small piece in a labeled Eppendorf tube. Keep the Eppendorf tube on dry ice. Repeat for all samples, using a different petri dish and surgical blade for a new sample.

SECTION: Isolation of Human Brain Nuclei
2. Optional: Weigh Eppendorf tubes if needed and record weights. 50 mg per sample.

SECTION: Isolation of Human Brain Nuclei
3. Pour 25 mL per sample of sucrose homogenization buffer into 50 mL Falcon tube and place on ice. Add 1 µL of RNase Inhibitor (40 U/μL) per mL of homogenization buffer. Shake tube to mix.

SECTION: Isolation of Human Brain Nuclei
4. Pipette 15 mL homogenization buffer with RNase Inhibitor into 15 mL Dounce tissue grinder on ice. Add tissue piece and homogenize. 
 

SECTION: Isolation of Human Brain Nuclei
5. Label bottom of 50-mL ultracentrifuge tube (in case it breaks in the centrifuge). Pipette 10 mL fresh homogenization buffer to the tube as a cushion, and carefully pour the 15 mL of homogenate on top of it to maintain two separate layers.

SECTION: Isolation of Human Brain Nuclei
6. Place the ultracentrifuge tubes in the swing buckets. Weigh and balance the buckets containing the samples by adding a few drops of fresh homogenization buffer to the top.
SECTION: Isolation of Human Brain Nuclei
7. Ultracentrifuge at 25000 rpm, 60 min, 4 °C in SW28 rotor for ultracentrifuge. 
 

SECTION: Isolation of Human Brain Nuclei
8. Add 2 µL RNase Inhibitor (40 U/μL) per mL of nuclei suspension buffer, making 5 mL total buffer per sample. Mix and place on ice.
SECTION: Isolation of Human Brain Nuclei
9. Remove all the supernatant by pouring off into a petri dish. Place upside-down in petri dish for a few seconds. Cut off the wall of the thin centrifuge tube with a clean razor blade.
SECTION: Isolation of Human Brain Nuclei
10. Resuspend the nuclei pellet at the bottom of the cut ultracentrifuge tube in 500 µL nuclei suspension buffer. Pipette gently up and down in the solution 10–20 times. Transfer this buffer into a labeled Eppendorf tube and repeat with an additional 500 µL nuclei suspension buffer. Place on ice.

SECTION: Isolation of Human Brain Nuclei
11. In a new labeled Eppendorf tube, make a 1:1 dilution of the nuclei suspension and Trypan Blue. Count nuclei (round blue dots) using haemocytometer slide (C-Chip, DHCN015). Count each 4x4 grid at the corners and average the counts.
SECTION: Isolation of Human Brain Nuclei
12. Concentration of the nuclei: Average count x 2 (dilution in Trypan Blue) x 104/mL.
SECTION: Isolation of Human Brain Nuclei
13. Spin down nuclei at 800 x g, 4 °C and 9 acceleration/deceleration for 10 min.
SECTION: Isolation of Human Brain Nuclei
14. Carefully remove supernatant with pipette.
 
 Resuspend in nuclei suspension buffer: the ideal concentration for 10X is 1000 nuclei/μL.
SECTION: Isolation of Human Brain Nuclei
15. Make a new 1:1 dilution of the nuclei suspension and Trypan Blue. Recount nuclei to ensure the concentration is adequate for 10x (700-1200 nuclei/μL). Ideally, there would be 50 nuclei per 4x4 grid in the haemocytometer slide.
SECTION: Isolation of Human Brain Nuclei
16. Submit samples for 10x Genomics Chromium loading (single cell 3’ V3.1 chemistry).