Choosing the right gene expression assay for your experiment
10x Genomics
Powering researchers’ insights with innovative single cell, spatial biology, & in situ tools. ????
Cell heterogeneity is a key contributor to biological complexity that is often masked by bulk techniques, such as RNA sequencing (RNA-seq) or microarray analysis. Rather than providing an average snapshot of how cells work, single cell RNA sequencing (scRNA-seq) technology gives researchers the ability to more fully characterize tissue heterogeneity, identify rare cell types, and dissect molecular mechanisms cell by cell.?
Figure 1: scRNA-seq reveals cellular heterogeneity that is masked by bulk RNA-seq methods.
This high level of resolution leads to groundbreaking insights across a variety of fields, as evidenced by over 3,200 publications (and counting) featuring Chromium technology from 10x Genomics. Scientists who wield this technology have identified new, rare cell types, delineated the molecular underpinnings of variable drug responses and toxicities, disentangled the complex cellular heterogeneity of tumors, and even developed a CRISPR-based map that assigned a function to every gene.
Uncovering the cellular diversity underlying complex biological systems—especially during disease development, progression, and treatment—is critical not only to answering novel scientific inquiries, but improving human health through the development of better diagnostics and drugs for complex diseases, such as cancer and neurodegenerative disorders.
Now that you know how scRNA-seq can elevate your research, where do you start? Below, we discuss the benefits of each of our single cell products to help you pick the one best suited to answer your exciting scientific questions.
When should I use the Single Cell Gene Expression Flex assay?
Single cell genomics has blown up over the past decade, but gaps in its capabilities have remained.
By and large, the use of single cell transcriptomics has come with logistical hurdles, most notably sample acquisition and processing. Working with fresh cells can be challenging and necessitates prompt sample preparation. Furthermore, accessing certain sample types can bring additional difficulties, with low-quality samples yielding damaged RNA, whole tissues requiring expertise in sample collection and preparation, and biobanked tissues lying out of reach without effective methods to extract intact cells or nuclei for analysis.
Chromium Single Cell Gene Expression Flex lets you leave these issues with sample acquisition and processing in the past. This assay works with nearly any starting sample type, including FFPE and PFA-fixed tissues, cells, or nuclei. Not only does this powerful assay give you the flexibility you need, it is more sensitive than our flagship Chromium Single Cell Gene Expression 3’ kit (though the 3’ kit might still be a better fit for some as detailed below).
Figure 2: scRNA-seq comparison data from the Fred Hutch Innovation Lab (@HutchInnovation) demonstrates that Chromium Single Cell Gene Expression Flex (10X FRP; green) is roughly two times as sensitive as the Chromium Single Cell Gene Expression 3’ kit (10X 3’; red). Additional data and details on experimental setup can be found in this thread from the Fred Hutch Innovation Lab.
Because of this, we recommend the Single Cell Gene Expression Flex assay for everyone running single cell experiments with human or mouse samples who is looking to measure gene expression alone or simultaneously with cell surface protein expression. Chromium Single Cell Gene Expression Flex is our most sensitive, cost-effective, and flexible assay.?
Key takeaways:
Learn more here.
When should I use other 10x gene expression assays?
Although Single Cell Gene Expression Flex is our new go-to scRNA-seq assay, you may need a more specialized kit to address your research inquiry. Our other kits are better suited to scientists using non-human or non-mouse samples or those who are interested in profiling the immune cell or chromatin accessibility landscape within their samples.
Single Cell Gene Expression
Our Chromium Single Cell Gene Expression 3’ kit is our recommendation for researchers profiling non-human or non-mouse samples, including rats (and even axolotls). You can also automate this assay on our Chromium Connect instrument, which minimizes handling and empowers users to generate consistent and reproducible single cell gene expression results across experiments, across users, and even across multiple sites.
Key takeaways:?
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Single Cell Multiome ATAC + Gene Expression
Transcriptomics is an important piece of the biological puzzle, but may not tell a cell's entire story. The transcriptome, after all, is a byproduct of a highly coordinated program of gene expression regulated by the epigenome—a map of chemical changes to DNA’s chromatin state that leave it open (and accessible) or closed (and inaccessible). Chromatin state impacts how DNA-binding proteins like transcription factors or RNA polymerase can interact with genomic DNA, meaning that epigenomic regulation not only informs developmental decisions, disease progression, and therapeutic response, but also often precedes transcriptional changes and can be used to unravel otherwise indistinguishable cell types.
Researchers can tackle questions about the epigenome with the assay for transposase-accessible chromatin (ATAC)—a method that surveys the physical structure of the genome by identifying regions of open chromatin. We offer two options for profiling chromatin: Chromium Single Cell ATAC and Chromium Single Cell Multiome ATAC + Gene Expression. We are going to focus on our Chromium Single Cell Multiome ATAC + Gene Expression technology because it has all the benefits of ATAC and allows you to simultaneously acquire traditional gene expression data from the same cell.
To capture both the transcriptomic and chromatin accessibility landscape in a sample, some researchers split their samples, processing half with scRNA-seq and half with scATAC-seq. Then, they infer which cell types correspond between the two datasets. However, this method can be heavily reliant on assumptions about the relationship between transcription start site, promoter location, and gene expression levels, as well as a priori information about epigenetic profiles of different cell types. Further, differences that arise from the potentially disparate workflows by which the two samples are processed may impact cell state and recovery.
Our Chromium Single Cell Multiome ATAC + Gene Expression technology marries our proven Chromium Single Cell 3’ Gene Expression technology with our Chromium Single Cell ATAC-seq technology—direct links can be made between transcriptomic and epigenomic profiles, no guesswork required. Researchers can capture both modalities simultaneously in the same single cells, leading to the potential discovery of new gene regulatory interactions and a better interpretation of epigenetic profiles with key expression markers.
Figure 3: The Single Cell Multiome ATAC + Gene Expression workflow allows researchers to access a unified view of transcription and the chromatin landscape by combining gene expression and ATAC-seq data from the same single cell with a simple, streamlined workflow.
Key takeaway:
Learn more here.?
Single Cell Immune Profiling
The immune system has the extraordinary responsibility to protect the body from pathogens and diseased cells. To perform these functions, immune cells have unique surface proteins, including T-cell receptors (TCRs), B-cell receptors (BCRs), and cluster of differentiation (CD) molecules, that distinguish them from other cells and drive their key functions. While traditional cytometry techniques are effective to identify and pool immune cells according to known surface markers, a rigorous understanding of immune cell functionality requires further analytical attention, including study of the transcriptome, novel surface proteins and receptors, and antigen specificity.
Chromium Single Cell Immune Profiling is a multiomic solution to immunology questions many other conventional techniques aren’t well-equipped to answer. It allows researchers to analyze full-length V(D)J sequences for paired B-cell or T-cell receptors, cell surface protein expression, antigen specificity, and/or CRISPR edits, along with gene expression, all from the same single cell.
Figure 4: Leverage the power of DNA barcoding, single cell microfluidics, and next-generation sequencing to simultaneously profile a combination of cell surface markers, mRNA, full-length TCR/BCR sequences, and antigen specificity, all from the same single immune cells, at scale.
Key takeaways:
Learn more here.
What now?
Whether you are a single cell newbie or a super-user, our comprehensive toolkit of single cell tools are here to help. Bring single cell technology to your lab with one of our kits, or expand your research by incorporating a new single cell tool.