TITLE: Nuclei Isolation for HMBA 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 2.892 mL Nuclear Isolation Media to a 5 mL eppendorf. Then add 60 uL protease inhibitor, 30 μL 10% Triton X-100, 15 uL RNase inhibitor, and 3 μL 100 mM DTT.
SECTION: Prepare Stock Solutions
1. Make 50 mL Nuclear Isolation Media by filling a 100 mL bottle with 25 mL of MilliQ water and then adding 500 uL 1M Tris, 1.25 mL 1M KCl, 250 uL 1M MgCl2, and 4.28 g Sucrose. Shake until sucrose is dissolved then fill to 50 mL with MilliQ water. Filter it using a stericup into another clean bottle. (4°C - 2 weeks)
SECTION: Prepare Fresh Solutions
5. Make 15 mL Blocking Buffer by adding 13.425 mL 1X PBS, 1.5 mL 10% BSA, and 75 uL RNase inhibitor (0.2 U/uL) to a 15 mL falcon tube.
SECTION: Prepare Stock Solutions
2. 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)

For a smaller stock, make 1 mL by combining 900 uL MilliQ water with 100 uL Triton-X 100. Filter with a FlowMi after vortex and incubation. 
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. Place your sample on dry ice. Tare a weigh boat or tube on the scale and weigh your sample.
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 a 40 um filter.
SECTION: Homogenization
10. Centrifuge at 900 g/rcf for 10 minutes at 4°C.
SECTION: Blocking Buffer &amp; Antibodies
12. The amount of Blocking Buffer we will need to resuspend our nuclei pellet with after centrifuging depends on the number of controls we will run. Each control tube will require 50 μL of nuclei suspension, and our sample needs to have 1,000 μL of nuclei suspension left over. In this case we are running two controls.

SECTION: Blocking Buffer &amp; Antibodies
11. Add 950 μL Blocking Buffer to each of your control tubes.
SECTION: Blocking Buffer &amp; Antibodies
13. After the cell homogenate is done centrifuging, discard the supernatant and resuspend the pellet in 1,100 μL of Blocking Buffer. Incubate for 15 minutes on ice. 
SECTION: Blocking Buffer &amp; Antibodies
14. Add 50 μL of nuclei suspension to each control tubes.
SECTION: Blocking Buffer &amp; Antibodies
15. Add the corresponding antibodies to the control tubes:
2 μL NeuN-PE (1/500) to the PE-only control
0.5 μL Olig2-Alexa 488 (1/2000) to the A488-only control
SECTION: Blocking Buffer &amp; Antibodies
16. Add both 2 μL NeuN-PE and 0.5 μL Olig2-Alexa 488 to the sample tube.
SECTION: Blocking Buffer &amp; Antibodies
17. Place all tubes in a rotator placed in a dark 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. Decant and resuspend in 999 uL blocking buffer. Consider adding an additional wash step here.
SECTION: Wash
22. Store tubes on ice until FACS. Bring DAPI stock and Blocking Buffer with you to FACS in case we have to dilute the sample to lower the event count.
SECTION: Wash
20. Add 1 uL of 0.1 mg/mL DAPI, a final concentration of 0.1 ug/mL, to each sample and control tube and invert to mix.
SECTION: Wash
21. Add 300 uL Blocking Buffer to falcon FACS tubes and label them A488+, PE+, and Double Negative. We will sort into these.
SECTION: Prepare Stock Solutions
3. Make 1 mL of 0.1 mg/mL DAPI by adding 100 uL of 1 mg/mL DAPI stock to 900 uL of milliQ water. (4C - 1 month)