Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.

Area of Science:

• Molecular Biology
• Genomics
• Neuroscience

Background:

• Single-cell RNA sequencing (RNA-seq) from tissues often involves cell dissociation that can alter transcriptomes.
• Previous methods using protease treatment at 30 °C are known to damage cellular integrity and gene expression profiles.

Purpose of the Study:

• To develop a protocol for nuclear RNA sequencing (RNA-seq) that minimizes damage to the transcriptome.
• To enable RNA-seq analysis from archived postmortem human brain tissue.
• To investigate biological features unique to the cell nucleus.

Main Methods:

• Isolation of nuclei from tissue homogenates at 4 °C to minimize damage.
• Sorting of nuclei using Fluorescence-Activated Cell Sorting (FACS).
• Construction of cDNA libraries using Smart-seq2 and Nextera XT for RNA-seq.

Main Results:

• The developed protocol yields nuclear transcriptomes from postmortem human brain tissue stored at -80 °C.
• This method allows for RNA-seq analysis from individual neurons within archived brain samples.
• The protocol enables the study of nuclear-specific features, including transcript enrichment and noncoding RNA precursors.

Conclusions:

• The nuclear RNA-seq protocol provides a robust method for analyzing transcriptomes from preserved tissues.
• This technique expands the accessibility of valuable brain archives for molecular research.
• The protocol facilitates the investigation of nuclear-specific biological insights.