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
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).