TITLE: Nuclei Isolation for FACS
AUTHORS: Lakme Caceres

[DESCRIPTION]
This protocol is for purifying nuclei for downstream 10X sequencing.

[GUIDELINES]
Keep tissue/nuclei on ice as much as possible.

[STEPS]
SECTION: Prepare Fresh Solutions
4. Make 3 mL Homogenization Buffer by adding 2.9 mL Nuclear Isolation Media (filtered via syringe) to a 5 mL eppendorf. Then add 3 μL 100 mM DTT and 30 μL 10% Triton X-100.
SECTION: Prepare Stock Solutions
1. Make 20 mL 10% BSA by combining 2 mL of BSA with 18 mL of MilliQ water in a 50 mL falcon tube. (4°C - 2 weeks)
SECTION: Prepare Stock Solutions
2. Make 250 mL Nuclear Isolation Media by filling a 250 mL bottle with 200 mL of MilliQ water and then adding 2.5 mL 1M Tris, 6.26 mL 1M KCl, 1.25 mL 1M MgCl2, and 21.45 g Sucrose. Shake until sucrose is dissolved then fill to 250 mL with MilliQ water. (4°C - 2 weeks)
SECTION: Prepare Fresh Solutions
5. Make 10 mL Blocking Buffer by adding 8.2 mL MilliQ water, 1 mL 10X PBS, and 800 μL 10% BSA to a 15 mL falcon tube and vortex.
SECTION: Prepare Stock Solutions
3. Make 20 mL 10% Triton X-100 by combining 18 mL MilliQ water with 2 mL Triton X-100 in a 50 mL tube. Vortex and then incubate at room temperature for 20 minutes. Filter it through a 0.22 μm filter with a syringe into a clean 50 mL tube. (4°C - 1 month)
SECTION: Homogenization
6. Clean dounce, scalpel, and forceps using MilliQ water, ethanol, RNase Zap, then MilliQ again. The red-tape forceps are for unfixed tissue. 
SECTION: Homogenization
7. Get tissue sample from -80°C freezer and place on dry ice. Carve out tissue on an ice block then weigh out approximately 60 mg on a tared weigh boat.
SECTION: Homogenization
8. Add tissue to dounce and push it to the bottom using 1 mL of Homegenization Buffer and the pestle. Homogenize the tissue without creating bubbles. Then add the remaining 2 mL of the Homogenization Buffer and continue to dounce until homogenized.
SECTION: Homogenization
9. Pass all of the nuclei suspension through three FlowMi filters, 1000 μL at a time into a new 5 mL eppendorf. 

SECTION: Homogenization
10. Centrifuge at 900 g/rcf for 10 minutes at 4°C.
SECTION: Blocking Buffer &amp; Antibodies
11. Determine how many controls you will be using. Typically we have four tubes:

 
The amount of Blocking Buffer we will need to resuspend our nuclei pellet with after centrifuging depends on the amount of tubes we need. Each tube will require 50 μL of nuclei suspension, and our sample needs to have 1,000 μL of nuclei suspension left over. So in this case we will need    of Blocking Buffer for resuspending the nuclei pellet.

SECTION: Blocking Buffer &amp; Antibodies
12. Add 930 μL Blocking Buffer to each of the control tubes.
SECTION: Blocking Buffer &amp; Antibodies
13. After the cell homogenate is done centrifuging, discard the supernatant and resuspend the pellet in 1200 μL of Blocking Buffer. Incubate for 15 minutes on ice. 
SECTION: Blocking Buffer &amp; Antibodies
14. Add 50 μL of nuclei suspension to each of the four control tubes.
SECTION: Blocking Buffer &amp; Antibodies
15. Add the corresponding antibodies to the control tubes:
0.5 μL NeuN-PE
0.4 μL Olig2-Alexa 488
0.5 μL IgG-PE
0.5 μL IgG-Alexa 488
SECTION: Blocking Buffer &amp; Antibodies
16. Add 0.5 μL NeuN-PE and 0.4 μL Olig2-Alexa 488 to the sample tube.
SECTION: Blocking Buffer &amp; Antibodies
17. Place all tubes in a rotator placed in a 4°C fridge and incubate for 30 minutes.
SECTION: Wash
18. After incubation, spin down all of the tubes at 500 g for 5 minutes at 4°C. 
SECTION: Wash
19. If there is no visible pellet after centriguation, remove 950 μL of supernatant with a small pipette being careful not to disturb the bottom of the tube. Then resuspend the pellet in 950 μL Blocking Buffer.

If there is a visible pellet, remove all of the supernatant without disturbing the pellet and then resuspend in 930 μL of Blocking Buffer.
SECTION: Wash
22. Store tubes on ice until FACS. Bring Hoechst stock with you to FACS.
SECTION: Wash
20. Add 20 uL Hoechst to each sample and control tube and gently mix by tapping.
SECTION: Wash
21. Transfer all samples and controls to 2 mL tubes that will be less forceful on nuclei during FACS.