Prepare the 2X salt-Tris solution and aliquot to 1 ml tube and freeze at -20°C

Prepare the BSA in PBS and filter through 0.2um filter.

Prepare the Tween-20 Homogenization lysis buffer fresh for each preperations

Filter sucrose 2M solution through 0.2um/0.45um filter

Prepare cOmplete protease inhibitor (50X) by dissolving a tablet in 1 mL of water.

Filter protease inhibitor solution through 0.2um filter

Place the homogenizer at -80°C at the start and leave 10 min before homgenization on ice.

Cut brain piece on dry ice (if a large section), then transfer immediately in homogenizer containing 250 uL of TST+NP Homogenization Lysis Buffer. Immediately add 500uL TST+NP Homogenization Lysis Buffer

Allow the tissue to thaw for 2 mins in the homogenization buffer.

Homogenize tissue with pestle A (10X), wait 1min, and with pestle B (10X).

Put the homogenate through 70µm cell strainer and place the falcon on ice. Incubate on ice for 5 mins. Rinse dounce and filter with 250uL TST+NP HB.

During the incubation (in about 4 mins), use LUNA-fl counter to assess the viability of the nuclei.

Transfer the contents from the 50 mL falcon to a 2 mL protein lo-bind tube.

Centrifuge @ 500xg for 5 mins. Discard the supernatant.

Resuspend the pellet in 200 uL of Wash Buffer 1, and transfer to a 2mL DNA lobind tube.

Add additional 320 uL Wash Buffer 1 to a final volume of 520 uL.

Add 520uL of Gradient medium to sample (Vf = 1040 uL).

Layer 770 uL of 29% Cushion in the ultracentrifuge tube.

Layer the sample on top of cushion using a P1000, without disturbing the cushion.

Optional: Check tube weight, adjust weight to counter differences.

Centrifuge at least 3,000 rcf in a swinging bucket, at 4°C for 20 minutes with brake off.

Remove supernatant, remove the lower supernatant with P200, leaving about 50-100 uL.

Gently resuspend nuclei in ultracentrifuge tube and transfer to 1.5 mL DNA lobind tube.

Rinse ultracentrifuge tube with Wash buffer 2 and transfer also to 1.5 mL DNA lobind tube.

Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.

Resuspend in 0.1x lysis buffer (200 uL) and gently pipetmix 5x

Incubate on ice for 2 min

Add Wash buffer 2 (1mL) & gently pipetmix 5x

Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.

Resuspend in 1X Diluted Nuclei buffer. Count