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

Abstract

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)

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 compl....

Materials

List of materials used in this article

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