Nuclei Isolation for HMBA FACS

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)

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)

Make 500 uL 10 ug/mL DAPI by adding 5 uL of 10 mg/mL DAPI stock to 495 uL of 1X PBS.

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.

Make 15 mL Blocking Buffer by adding 13.425 mL 1X PBS, 1.5 mL 10% BSA, and 75 uL RNase inhibitor to a 15 mL falcon tube.

Clean dounce, scalpel, and forceps using MilliQ water, ethanol, RNase Zap, then MilliQ again. The red-tape forceps are for unfixed tissue.

Place your sample on dry ice. Tare a weigh boat or tube on the scale and weigh your sample.

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.

Pass all of the nuclei suspension through a 40 um filter.

Centrifuge at 900 g/rcf for 10 minutes at 4°C.

Add 950 μL Blocking Buffer to each of your control tubes.

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.

After the cell homogenate is done centrifuging, discard the supernatant and resuspend the pellet in 1100 μL of Blocking Buffer. Incubate for 15 minutes on ice.

Add 50 μL of nuclei suspension to each control tubes.

Add the corresponding antibodies to the control tubes:

• 0.5 μL NeuN-PE (1/2000) to the PE-only control
• 0.33 μL Olig2-Alexa 488 (1/3000) to the A488-only control

Add both 0.5 μL NeuN-PE and 0.33 μL Olig2-Alexa 488 to the sample tube.

Place all tubes in a rotator placed in a dark 4°C fridge and incubate for 30 minutes.

After incubation, spin down all of the tubes at 500 g for 5 minutes at 4°C.

If there is no visible pellet after centriguation, remove 980 μL of supernatant with a small pipette being careful not to disturb the bottom of the tube. Then resuspend the pellet in 980 μL Blocking Buffer.

If there is a visible pellet , remove all of the supernatant without disturbing the pellet and then resuspend in 980 μL of Blocking Buffer.

Add 20 uL 10 ug/mL DAPI to each sample and control tube and invert to mix.

Add 300 uL Blocking Buffer to falcon FACS tubes and label them A488+, PE+, and Double Negative. We will sort into these.

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.