Izolacja i multipleksowanie jądra o niskim poziomie wejściowym z przeciwciałami kodowymi zwojów współczulnych myszy do sekwencjonowania RNA pojedynczego jądra

Overview

This protocol outlines a low-input sample preparation method for single-nucleus sequencing, focusing on cardiac sympathetic neurons. It includes detailed steps such as dissection, cell dissociation, and nucleus isolation.

Key Study Components

Area of Science

• Neuroscience
• Single-nucleus sequencing
• Cardiac sympathetic neurons

Background

• Single-nucleus RNA sequencing allows for the analysis of neuronal samples.
• This method is particularly useful for studying the biological architecture of neurons.
• Challenges exist in sequencing larger neurons from human ganglia.
• Barcoding with hashtag oligos enables sample multiplexing.

Purpose of Study

• To characterize cardiac sympathetic neurons in health and disease.
• To develop a protocol for low-input sample preparation.
• To facilitate the sequencing of neuronal nuclei.

Methods Used

• Droplet-based single-nucleus RNA sequencing.
• Dissection of mouse superior cervical and stellate ganglia.
• Cell dissociation and cryopreservation techniques.
• Nucleus isolation and hashtag barcoding for multiplexing.

Main Results

• Successful isolation of nuclei from cardiac sympathetic neurons.
• Demonstration of the protocol by research technicians and PhD students.
• Potential extension of the method to human ganglia neurons.
• Enhanced understanding of neuronal architecture in various conditions.

Conclusions

• The protocol provides a reliable method for single-nucleus sequencing.
• It opens avenues for future research on human neuronal samples.
• Barcoding techniques improve the efficiency of sample analysis.

Frequently Asked Questions