Single Cell Sequencing Technology

What is Nucleic Acid Sequencing?

• Nucleic acid sequencing is a tool that yields information on the order of nucleotides (adenine, thymine, cytosine, and guanine) in DNA or RNA.
• Knowing the sequence helps us to know the genetic instructions required for life.

What is Bulk Sequencing?

• Bulk sequencing is the sequencing of the genome/transcriptome of a cell population, such as a tissue, organ, or entire organism.
• Its output is the "average genetic/transcriptomic" of the cell population.
• The major disadvantage of bulk sequencing is that it assumes cells from a given tissue are homogeneous, missing important cell-to-cell variability.

Bulk vs Single-cell RNA Sequencing

• Bulk RNA-seq mainly reflects the averaged gene expression across thousands of cells.
• Averaging that occurs in pooling large numbers of cells does not allow a detailed assessment of the fundamental biological unit—the cell—or the individual nuclei that package the genome.
• Single-cell RNA sequencing assesses the differences in gene expression between individual cells, identifying rare populations that cannot be detected from an analysis of pooled cells.

Single-cell Sequencing

• The main steps for single-cell sequencing are single cell isolation from cell populations, library preparation, sequencing of the library using a next-generation sequencer, and data analysis and visualization.

Single Cell Isolation from Cell Populations

• Single-cell isolation is the first step for obtaining transcriptome information from an individual cell.
• Cells can be isolated using different methods, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), laser capture microdissection, and manual cell picking or micromanipulation.

Fluorescence-Activated Cell Sorting (FACS)

• FACS is a widely used strategy for isolating highly purified single cells.
• In FACS, cells are first tagged with a fluorescent monoclonal antibody, which recognizes specific surface markers and enables sorting of distinct populations.
• The workflow of FACS involves the physical separation of particles, where cells are ejected into air, forming droplets that are then charged and deflected to the left or right of the original stream based on their fluorescence intensity and scattering properties.

Learn about single cell sequencing, its applications, and research areas, including bulk sequencing, RNA sequencing workflow, and nucleic acid sequencing.