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Quick Mastery of Single-Nuclear RNA Sequencing

   |  June 24, 2024

Single-Cell Sequencing (SCS) is a crucial tool in life sciences research, allowing scientists to study the genome, transcriptome, and epigenome at the single-cell level. Its significance lies in its ability to uncover subtle differences between cells that might be overlooked using traditional multicellular sequencing techniques.

In biomedical research, single-cell sequencing has extensive applications, covering areas such as cancer research, developmental biology, neuroscience, and immunology. Additionally, it has played a crucial role in COVID-19 research, aiding scientists in understanding how the virus affects different cell types and the host cell’s immune response. Single-cell sequencing helps researchers gain a deeper understanding of cellular complexity and the functional mechanisms of organisms.

Single-Nuclear RNA Sequencing

Despite the promising potential of single-cell sequencing in biomedical research, there are still several limitations to address. The results of single-cell sequencing heavily depend on the successful isolation of individual cells from complex tissue structures during sample preparation and cell separation. This requires high cell viability and sufficient cell numbers from dissociated tissue suspensions, making most valuable clinical samples processed via cryopreservation unsuitable for single-nucleus RNA sequencing (snRNA-seq).

Various organs and tissues are rich and diverse, with differences.

It is worth noting that single-nucleus RNA sequencing (snRNA-seq) has several significant advantages over traditional single-cell RNA sequencing (scRNA-seq).

  • 1. Broad Sample Applicability:

snRNA-seq is suitable for both fresh and difficult-to-dissociate or frozen samples, enabling the use of valuable clinical samples stored in ultra-low temperature freezers.

  • 2. Reduced Transcriptional Bias:

As nuclear transcriptional activities are suppressed and fixed during freezing, snRNA-seq can more accurately reflect the transcriptional state of cells, improving data reliability.

  • 3. Increased Cell Type Comprehensiveness:

snRNA-seq extracts nuclei directly from frozen states via mechanical or chemical disruption, avoiding dissociation biases introduced by enzymatic methods, thus more comprehensively recovering cell types.

  • 4. Relatively Simple Procedures:

Nuclei extraction is simpler compared to preparing single-cell suspensions, reducing potential nuclear loss.

  • 5. Enhanced Cell Type Identification Resolution:

snRNA-seq enhances the sensitivity of cell type identification by analyzing nuclear RNA, including intronic reads.

Overall, snRNA-seq is a powerful tool that addresses some of the major issues found in scRNA-seq.

RWD offers solutions for single-nucleus extraction from tissue and library pre-construction.

Preprocessing of single-cell sequencing

Tissue Sample Dissociation:

RWD Single Cell Suspension dissociator can be set up with one click, using an integrated nucleus extraction program for precise mechanical cutting. This allows for the dissociation of tissue samples from various sources, efficiently and rapidly obtaining single-nucleus suspensions from fresh and frozen tissue samples. Cut the tissue into small pieces, place them in RWD tissue processing tubes with mixed reagents, run the preset nucleus extraction program for dissociation, then perform ice reaction lysis, and collect the single-nucleus suspension.

Nuclear staining with trypan blue under microscopy, showing cell debris and other impurities <10%
Nuclear staining with trypan blue under microscopy, showing cell debris and other impurities <10%

Single-Nucleus RNA-seq:

Prepare the single-nucleus suspension, ensuring quality control for subsequent sequencing requirements. The sequencing steps generally include separating nuclei into individual units, adding reagents to encapsulate them into droplets/beads, followed by a series of reverse transcription, cDNA amplification, and library construction processes. Finally, sequence the library using an appropriate platform and analyze the data to obtain sequencing results.

In conclusion, single-nucleus sequencing technology has advantages over single-cell RNA sequencing in terms of sample applicability. The preparation of single-nucleus suspensions is simpler compared to single-cell suspensions, and the genetic information obtained is relatively richer. However, when deciding which sequencing method to use, decisions should be based on the actual situation, as single-nucleus RNA sequencing cannot completely replace single-cell RNA sequencing. The application of single-nucleus RNA sequencing still requires ongoing exploration and research.

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