While single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity, it represents just one facet of the burgeoning field of single-cell technologies. This module delves into other powerful techniques that allow us to probe the genome, epigenome, proteome, and even the spatial organization of individual cells, offering complementary insights into biological systems.

Beyond RNA, researchers can now analyze the DNA of individual cells. This includes techniques like single-cell whole-genome sequencing (scWGS) to detect genomic alterations (mutations, copy number variations) at an unprecedented resolution, crucial for understanding cancer evolution and developmental abnormalities. Single-cell ATAC-seq (scATAC-seq) is another key technology, revealing the accessible regions of the genome, which are indicative of gene regulatory activity, without needing to sequence the entire genome.

The epigenome, encompassing modifications like DNA methylation and histone modifications, plays a critical role in gene regulation and cell identity. Technologies like single-cell bisulfite sequencing (scBS-seq) allow for the genome-wide profiling of DNA methylation at single-cell resolution. Similarly, single-cell CUT&RUN and CUT&Tag assays can map histone modifications and transcription factor binding sites in individual cells, providing a detailed understanding of how epigenetic states dictate cellular function and differentiation.

While RNA levels can be a proxy for protein abundance, direct measurement of proteins in single cells offers a more direct view of cellular function. Techniques like mass cytometry (CyTOF) and highly multiplexed immunofluorescence allow for the simultaneous detection of dozens of proteins within individual cells. Newer methods are emerging that integrate proteomic analysis with other single-cell modalities, providing a multi-omic picture.

Understanding where molecules are located within a tissue is as important as knowing their abundance. Spatial transcriptomics technologies preserve the spatial context of RNA molecules, allowing researchers to map gene expression patterns within intact tissue sections. Similarly, spatial proteomics techniques are emerging to map protein distributions. These methods are revolutionizing fields like developmental biology, neuroscience, and cancer research by revealing how cellular interactions and tissue architecture influence function.

The ultimate goal is often to integrate information from multiple molecular layers within the same cell. Multi-omic single-cell technologies are rapidly advancing, enabling simultaneous measurement of, for example, RNA, DNA, and protein, or RNA and chromatin accessibility, from individual cells. This holistic approach provides a more comprehensive understanding of cellular states and regulatory mechanisms.

The selection of a single-cell technology depends heavily on the research question. Are you interested in gene expression dynamics, regulatory potential, protein function, or spatial organization? Understanding the strengths and limitations of each platform is crucial for designing effective experiments and interpreting results accurately.