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

Overview

This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Protocol

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

Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma	M6250
CaCl2	Sigma	21115-100ML
EDTA (0.5 M)	Thermo Scientific	R1021
Falcon 15 mL Conical Centrifuge Tubes	Fisher Scientific	352096
MACS SmartStrainers (100 µm)	Miltenyi Biotec	130-098-463
Mg(Ac)2	Sigma	63052-100ML
NP-40	Abcam	ab142227
Nuclei Isolation Kit: Nuclei EZ Prep	Sigma	NUC101-1KT
Safe lock tubes 1.5 mL	Eppendorf	30120086
Safe lock tubes 2.0 mL	Eppendorf	30120094
Sucrose	Sigma	S0389
Wide Bore pipette tips (1000 µL)	Themo Fisher Scientific	2079GPK