Nuclei Isolation for 10x Chromium single-nuclei RNA sequencing

Prepare ~50mLof ice-cold PBS and store it on ice1. Prepare 8mLof ice-cold lysis buffer containing 0.2U/ul of RNase inhibitor by adding 40µL of RNase inhibitor to 8mLof Nuclei EZ-prep

1. Prepare 10mLof ice-cold wash buffer containing 1%BSA and 0.2U/ul of RNase inhibitor by adding 2mLof 5%BSA and 50µL of RNase inhibitor to 8mL of 1xPBS

Perfuse the animal with ice-cold PBS1. Rapidly and On ice, extract the brain and using fine forceps, dissect the rat striatum containing the human xenograft

1. Immediately transfer the tissue in an ice-cold 1.5mL tube and snap-freeze in in liquid nitrogen or dry ice
2. Store the sample to -80°Cuntil use

On ice Transfer the tissue from the -80°C to the dounce homogenizer containing 1.5mL of cold lysis buffer On ice1. Homogenize with 24 strokes On ice

1. Transfer the homogenized sample to a 15ml Corning tube on ice
2. Wash the dounce with 1.5mLof lysis buffer
3. Allow the tube to stand for 0h 5m 0s onOn ice
4. Centrifuge at 500x g,0h 0m 0s for 0h 5m 0s at 4°C
5. Remove the supernatant
6. Wash with 1mLof lysis buffer on ice
7. Wait 0h 5m 0s on On ice
8. Centrifuge at 500x g,0h 0m 0s for 0h 5m 0s at 4°C
9. Remove the supernatant
10. Resuspend in 1mL wash buffer (gently, without creating bubbles) On ice
11. Filter with a 30um cell strainer (MACS SmartStrainers)On ice
12. Wash the strainer with 1mL wash buffer On ice
13. Centrifuge at 500x g,0h 0m 0s for 0h 5m 0s at 4°C
14. Remove the supernatant
15. Resuspend in300µL wash buffer On ice
16. Count the nuclei. We recommend using both Trypan Blue to assess nuclei integrity and a fluorescent cell counter When looking at the nuclei in the counter, look at the integrity of them – how round they are with clear border and how much they are single and dispersed and not in clumps or in small groups of cells
17. Stain using HNA-PE and NeuN-Rb (1:100 diluted in wash buffer)
18. Incubate for 0h 30m 0s at 4°C in the dark
19. Add secondary antibody (Rb-647, 1:200)
20. Incubate for 0h 20m 0s at 4°C in the dark
21. Wash with 1mL of wash buffer On ice
22. Centrifuge at 500x g,0h 0m 0s for 0h 5m 0s at 4°C
23. Wash with 1ml of wash buffer 0h 20m 0s
24. Centrifuge at 500x g,0h 0m 0s for0h 5m 0s at 4°C
25. Resuspend in DAPI solution (dilute the stock of 1mg/ml in 1:1000, 1ul in 1ml)
26. FACS sort the nuclei in 35µL of wash buffer using a 70 μm nozzle, 21– 22 p.s.i. The sort should be done for HNA⁺NeuN⁺ DAPI⁺ single nuclei
27. After FACS sorting, centrifuge the nuclei 600x g,0h 0m 0s for 0h 8m 0s
28. Carefully remove as much supernatant as possible and count in a cell counter. Again, look at the integrity of the nuclei. The nuclei should be counted twice and the average concentration calculated.
29. The nuclei are now ready for a 10x run. In our case sample processing was performed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomic, California, # PN-1000121) and the Chromium Controller (10X Genomics, California) per manufacturer's instructions as published in User Guide CG000204 Rev D (10X Genomics,California)