PUBLICATION
Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method
- Authors
- Eski, S.E., Dubois, C., Singh, S.P.
- ID
- ZDB-PUB-200715-9
- Date
- 2020
- Source
- Journal of visualized experiments : JoVE (160): (Journal)
- Registered Authors
- Singh, Sumeet Pal
- Keywords
- none
- MeSH Terms
-
- Analytic Sample Preparation Methods
- Animals
- Brain/cytology
- Cell Fractionation/methods*
- Cell Nucleus*/drug effects
- Cells, Cultured
- Detergents/pharmacology*
- Flow Cytometry
- Gene Expression Profiling
- High-Throughput Nucleotide Sequencing
- Reproducibility of Results
- Zebrafish
- PubMed
- 32658191 Full text @ J. Vis. Exp.
Citation
Eski, S.E., Dubois, C., Singh, S.P. (2020) Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method. Journal of visualized experiments : JoVE. (160):.
Abstract
High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping