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

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.

Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

“…To isolate nuclei from human islet preparations, we tested the Nuclei EZ lysis buffer-based protocol. We employed this isolation method because it was previously successfully used to isolate nuclei from frozen compact tissues, such as tumor tissues [ 58 , 59 ]. In our hands, we validated that this protocol removes cytoplasmic content quickly and consistently.…”

Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas

“…The nuclei used for snATAC-seq were paired with snRNA-seq samples. 18 For annotation, we performed label transfer from snRNA-seq to snATAC-seq data, excluding the gradient population that did not fit any NMF metaprogram. We identified NMF-derived tumor and TME populations in oligodendrogliomas ( Figure 2 D) and astrocytomas ( Figure 2 E).…”

A protocol for single nucleus RNA-seq from frozen skeletal muscle

“…Common elements that vary between protocols include buffers, instruments of dissociation, and methods of purification. This is emphasized by the distinct protocols developed to isolate nuclei from the liver (Cavalli et al, 2020), kidney (Leiz et al, 2021), and tumours (Narayanan et al, 2020;Slyper et al, 2020). We have developed a protocol that works for the dense, fibrous nature of skeletal muscle.…”