Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

1. Tissue Dissection and Dissociation

1. Transfer fresh frozen tissue sample (10–60 mg) to a pre-chilled Petri dish. Mince/chop fresh frozen tissue with a razor blade to small pieces on ice.
2. Add 500 μL of chilled nuclei lysis buffer to a pre-chilled 1.5 mL tube. Place the tissue pieces in the 1.5 mL tube containing the nuclei lysis buffer and transfer to a Douncer (Table of Materials).
   NOTE: There are two Dounce homogenizer pestles: “loose” or “A” pestle for initial sample reduction and “tight” or “B” pestle for complete sample homogenization.
3. Dounce the tissue pieces with the “loose” pestle for about 20 strokes, until friction is reduced. If debris is present, the sample may be pre-cleared by filtration with a 100 mm strainer.
4. Dounce with the “tight” pestle for 20 strokes to achieve complete tissue homogenization.
5. Transfer the homogenate (about 500 μL) into a pre-chilled 2 mL tube. Add 1 mL of chilled lysis buffer. Mix gently and incubate on ice for 5 min. Gently mix with a wide-bore pipette tip and repeat 1–2 times during the incubation.
6. Filter the entire homogenate using a 30 μm strainer mesh, collect into a 15 mL Falcon tube and transfer back into a new pre-chilled 2 mL tube. A single strainer is typically sufficient for the entire homogenate.
7. Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round and the nuclear membrane should not be distorted. If debris is present, nuclei can be re-filtered.
8. Centrifuge the nuclei at 500 x g for 5 min at 4 °C on a benchtop centrifuge. Remove the supernatant, leaving behind ~50 μL with a pellet containing the nuclei. Gently resuspend the pellet in another 1 mL of nuclei lysis buffer and incubate for 5 min on ice.
9. Centrifuge the nuclei at 500 x g for 5 min at 4°C. Remove the supernatant without disturbing the pellet, add 500 mL of 1x homogenization buffer (HB) (Table 1) and incubate for 5 min without resuspending. Then, resuspend the nuclei in another 1.0 mL of 1x HB.
10. Centrifuge the nuclei at 500 x g for 5 min at 4 °C. Remove the supernatant and gently resuspend the nuclei in 200 μL of 1x HB into a new 2.0 mL tube.

    Table 1: Preparation of 1x Homogenization Buffer Unstable Solution (2 mL per sample)

    1x Homogenization Buffer Unstable Solution (2 mL per sample)
    Reagent	Final Conc.	Vol per sample (μL)
    6x Homogenization Buffer Unstable	1x	333.33
    1 M Sucrose	320 mM	640.00
    50 mM EDTA	0.1 mM	4.00
    10% NP40	0.1%	20.00
    H2O		1006.27