Isolation of trigeminal neural nuclei

1. Cut tissues into small pieces (1-2 mm) and incubate in RNAlater (ThermoFisher, cat# AM7021) overnight at RT or 4℃
2. Remove excess RNAlater and freeze tissue pieces in a sterile microfuge tube on dry ice. Store at -80℃ until use
3. On day of 10x run:  prepare homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris, pH 8.0, 1 µM DTT, 0.1% Triton X-100 (v/v).  and cool on ice.
4. Cool down all necessary equipment and solutions
5. Homogenize tissues in glass dounce homogenizer (Fisher Scientific, Cat# 357538) in 1ml of cold homogenization buffer (make fresh) ~5 strokes with “loose” pestle and ~10 strokes with “tight” pestle … on ice
6. Filter through 40 µm cell strainer (ThermoFisher Scientific, cat# 08-771-1)
7. Transfer to microfuge tube (low bind; Sorenson BioScience, cat# 11700) and spin @ ~300-800g at 4° for 5 mins
8. Remove supernatant and resuspend pellet in 500 µl of PBS + 1% BSA + SUPERaseIn RNase Inhibitor ( 0.2 U/µ; ThermoFisher Scientific, Cat# AM2696)
9. Incubate on ice for 10-15 mins
10. Add rabbit polyclonal anti-NeuN antibody (Millipore, cat#ABN78) 1:2000 to 1:5000 or antibody of choice.
11. Incubate with rotation at 4° for 30 mins
12. Spin @ ~300-800g at 4° for 5 mins
13. Remove supernatant and “wash” by adding 1 ml PBS + 1% BSA + SUPERaseIN RNase inhibitor
14. Spin @ ~300-800g at 4° for 5 mins
15. Resuspend pellet in 80 µl of PBS + 0.5% BSA + 2mM EDTA
16. Add 20 µl of anti-rabbit IgG Microbeads (Miltenyi Biotech, cat# 130-048-602).  Volume is for 10⁷ total cells.  Adjust volumes in steps 15 and 16 accordingly if you have more total cells.
17. Incubate at 4° 15-20 mins
18. "wash" by adding 1-2 ml of PBS + 0.5% BSA + 2mM EDTA
19. Spin @ ~300-800g at 4° for 5 mins
20. Remove supernatant and resuspend in 0.5-1 ml PBS + 0.5% BSA + 2mM EDTA
21. Load onto LS column (Miltenyi Biotec, cat# 130-042-401) – follow instruction from manufacturer:
    1. put column on magnetic MidiMACS separator (Miltenyi Biotec, cat#130-042-302; MACS MultiStand (cat#130-042-303), add ice cold 3 ml of buffer (PBS + 0.5% BSA + 2mM EDTA) to equilibrate
    2. resuspend nuclei pellet (from step 20 above) in 0.5-1 ml buffer (1X PBS + 0.5% BSA + 2mM EDTA)
    3. Add nuclei to column
    4. Wash column with 3 ml  ice cold buffer (3x)
    5. Remove column from magnet and elute in 5 ml of same buffer using plunger that comes with the LS column. Elute on ice into 15 ml Falcon tube.
22. Spin @ 500g at 4° for 10 mins
23. Remove supernatant 
24. Resuspend pellet in 1-1.5 ml of PBS + 1% BSA + SUPERaseIn RNase Inhibitor
25. Check for nuclei clumping.  If there are any, then use Ultra-Turrax (step 26) or another method to remove clumps. If not, then count nuclei with a hemocytometer.
26. Homogenize with Ultra-Turrax (Laboratory Supply Network, Inc., cat#IKA:3737001) on setting 1 for no more than 45 sec on ice (adjust time accordingly … depending on many nuclei you have).  You may have to increase the volume. 
    1. 1^st count:  Stain 5-10 µl with trypan blue and count (using a hemocytometer) also check for clumps
27. Transfer to microfuge tube and spin @ ~300-800g at 4° for 5 mins
28. Remove supernatant and resuspend in desire volume with PBS + 1% BSA + SUPERaseIn RNase Inhibitor … volume based on the first count. 
29. Usually I try to go for 1000 nuclei/µl (if there are enough nuclei).  I also assume that the first count is not that accurate (too diluted), so usually resuspend nuclei in ½ the calculated volume.
30. Stain 5-10µl with trypan blue and do a 2^nd count
31. Optional:  do a 3^rd count.  This should be close to the 2^nd count.
32. Check for nuclei quality under 40-60x bright field.

Homogenization Buffer:

250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris, pH 8.0, 1 µM DTT, 0.1% Triton X-100

** Can also use EZ PREP buffer (Sigma, Cat #NUC-101) for the homogenization buffer.

Protocol described in Nguyen et al (2019) Stereotyped transcriptomic transformation of somatosensory neurons in response to injury eLife 8:e49679