Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Summary

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Abstract

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.

Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Introduction

Diffuse lower grade gliomas (LGG), the most common primary brain tumor in adults, are infiltrative neoplasms that often arise in the cerebral hemisphere. LGGs exhibit both inter- and intra-tumor heterogeneity, which is not only driven by the tumor population but also by the non-malignant cells intricately involved in tumor development and progression^1,2,3,4,5.

Over the last decade, there has been an avalanche of genomic data gathered in the field of gliomas. These data mainly com....

Protocol

Fresh frozen glioma samples were obtained from the National Center for Tumor diseases (NCT)-tissue bank in Heidelberg, Germany. The use of patient material was approved by the Institutional Review Board at the Medical Faculty of Heidelberg, and informed consent was obtained from all patients included in the study.

1. Experimental preparation

1. Perform all steps on ice or at 4 °C.
2. Pre-chill tubes, dishes, razor blades, Douncers and pestles to 4 °C.
3. Prepare all buffers in advance. These buffers are stable at room temperature. Sterile filtration is recommended, especially for sucrose. The stock preparati....

Results

Single nucleus genomics is an evolving field with limited data and protocols. A critical factor that influences the outcome of single nuclei assays is the isolation of pure and intact nuclei. We combined two published protocols (DroNc-seq and Omni-ATAC-seq protocols) to isolate high-quality and pure nuclei from fresh frozen glioma tissue blocks in a relatively short time thereby maintaining the stability of the transcripts (Figure 1).

The use of various filtr.......

Discussion

The field of intra-tumoral heterogeneity is at an exciting stage, with novel assays and platforms being developed to challenge and expand the existing knowledge. Intra-tumoral heterogeneity is a crucial factor that contributes to disease progression and resistance to current treatment modalities in gliomas²⁸. Recent studies on brain tumors have focused on this important aspect by using single cell transcriptomic and epigenomic assays to better characterize the cellular heterogeneity within the sam.......

Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma	M6250
CaCl2	Sigma	21115-100ML
Dounce Homogenizer	Active motif	40401
EDTA (0.5 M)	Thermo Scientific	R1021
Falcon 5 mL Round Bottom Polystyrene Test Tube	Corning	352235
Iodixanol (aka Optiprep)	Stem cell technologies	07820
MACs Smart Strainers (30 µm)	Miltenyi Biotec	130-098-458
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
Phenylmethanesulfonyl fluoride (PMSF)	Sigma	P7626
Pre-Separation Filters (20 µm)	Miltenyi Biotec	130-101-812
Safe lock tubes 1.5 mL	Eppendorf	0030120086
Safe lock tubes 2.0 mL	Eppendorf	0030120094
Single Cell ATAC	10x Genomics
Single Cell Gene Expression	10x Genomics
Sucrose	Sigma	S0389
Wide Bore pipette tips (1000 µL)	Themo Fisher Scientific	2079GPK
Wide Bore pipette tips (200 µL)	Themo Fisher Scientific	2069GPK