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

Lucas Stalder; Fabian Koechl; Kerstin Hahn; Marc Sultan; Megana K. Prasad

doi:10.3791/65611

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

JoVE Journal
Biology

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JoVE Journal Biology

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

3,867 Views

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07:12 min

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July 28, 2023

DOI:

10.3791/65611-v

DOI:

10.3791/65611-v

•

07:12 min

July 28, 2023

•

41 Views

Lucas Stalder¹, Fabian Koechl¹, Kerstin Hahn¹, Marc Sultan¹, Megana K. Prasad¹

¹Roche Pharma Research and Early Development, Pharmaceutical Sciences,Roche Innovation Centre Basel

Transcript

This research focuses on supporting drug discovery studies using single nuclei RNA sequencing to investigate the efficacy and safety of potential treatments. Therefore, we are developing standardized nuclei extraction protocols for fresh frozen tissue samples from various organs. Our goal was to automate and standardize nuclei extraction to achieve consistent and reliable results by reducing non-biological variability.

The advantage is the partial automation through the use of a robotic dissociator and the simple and short workflow that can be applied on various tissue samples of different species. Having standardized protocols can help us answer questions about drug biodistribution, as well as explore the mechanisms underlying any safety signals that we might observe. In the future, we will be testing the applicability of our protocol for other downstream applications, such as ATAC-seq or multiomics.

We will also try to establish protocols to detect virally expressed transgenes in single nuclei.

Summary

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.