Name: Video: Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing
Uploaded: 2023-07-28T13:30:00
Duration: 2 min 2 s
Description: 500 Views. Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing

isolation of single nuclei from frozen mammalian tissues for single nuclei rna sequencing

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

Single-cell (sc) and single-nucleus (sn) RNA sequencing have become commonly used protocols in molecular and cellular biology due to the increased resolution of gene expression compared to bulk RNA sequencing. However, the isolation of good quality single cell and single nuclei preparations from solid tissues remains a challenge and is often the rate-limiting step in sc/sn-RNAseq experiments. Indeed, a plethora of protocols has been developed that use various chemical and mechanical procedures to obtain cell/nuclei suspensions1,2,3,4,5,6,7,8,9,10,11,12,13,14,15. Furthermore, strategies to clean up such preparations from debris/clumps, etc., range from flow sorting to filtration to washing. Such protocols are often manual (leading to user-related variability), can be time-consuming (leading to reduced cell/nucleus viability), and/or may require access to a flow cytometer for cell/nucleus sorting. This&#160;study focused on developing a simple, quick, and partially automated single nuclei isolation protocol from frozen mammalian tissues for downstream RNA sequencing applications. We focused specifically on nuclei isolation as opposed to cell isolation as it is compatible with the use of frozen tissues, rendering sample collection/processing more practical and enabling unbiased batching of samples, especially in time-course experiments. Furthermore, although the nuclear transcriptome does not fully reflect the cellular transcriptome, several studies have now shown that single nuclei RNA sequencing data is comparable to single cell RNA sequencing data for cell-type identification, even though the proportions of cell types can vary6,16,17,18,19.
Nuclei isolation consists of several steps: 1) mechanical or chemical disruption of the tissue to release the nuclei, 2) clean-up of debris and clumps, and 3) accurate counting of nuclei for preparation for downstream applications. In a number of protocols, step 1 frequently involves the use of a Dounce homogenizer in order to disrupt the tissue3,20. Alternatively, chemical methods can be used, although these often need to be optimized for different tissues2,5,6. We have experienced that a manual tissue disruption procedure is prone to operator-associated variability, leading to variable quality and yield of nuclei. In order to minimize technical variability and to have a more consistent and reproducible protocol that works across tissues, a protocol that uses a commercially available robotic tissue dissociator was developed21. For step 2, although buffer exchange is usually the simplest means for washing nuclei, we adopted the use of a relatively short sucrose gradient centrifugation step to have a more thorough removal of debris. For brain tissue specifically, we use a silica colloid gradient instead of a sucrose gradient&#160;for more effective myelin removal. Finally, for counting, the use of a hemocytometer is the gold standard for counting and visually inspecting the nuclei. In our protocol, this step can be reliably automated using a commercially available automated fluorescent cell counter22. This protocol has been tested and is compatible with several frozen mammalian tissues, including brain, kidney, spleen, and liver, from different mammalian species (rat, mouse, and non-human primate) and provides good quality nuclei for downstream single nuclei RNA sequencing with a droplet-based commercial platform. The protocol takes approximately 75 min from tissue preparation to the start of the single nuclei RNA sequencing workflow.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Watch this Scientific Journal Video about Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing at JoVE.com

Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing  

isolation-single-nuclei-from-frozen-mammalian-tissues-for-single

Research

JoVE Journal

Biology

500 Views. Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing

Video: Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing  

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.