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

Published: April 30, 2023

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 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. 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 homog…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples. J. Vis. Exp. (Pending Publication), e20616, doi: (2023).

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