Sep 6, 2022 / Oncology / Neuroscience / Immunology / Developmental Biology / Software

Single cell RNA-seq: An introductory overview and tools for getting started

Sheila Clark

Since the publication of this blog, we have introduced two powerful additions to our Chromium Single Cell technology platform: improved versions of our 3' and 5' RT-based assays (now powered by GEM-X) and the Flex probe-based assay for profiling of fresh, frozen, or fixed samples. Learn more about the next generation of single cell technology!

Swimming in a sea of cells: Why single cell genomics?

Complex biological systems result from the coordinated functions of individual cells performing an intricate "dance," each contributing their own special role to the ensemble. Because of this complexity, gene expression research in organisms, tissues or cell populations is often limited by traditional bulk RNA-seq methods. As scientists, we intuitively understand that when we "grind and find" in order to perform experiments on our diluted mix of RNA, we are really only getting a small idea of what might be going on in our cells of interest: cell-to-cell differences are missed and cellular heterogeneity can be completely masked. In fact, bulk RNA expression analysis often describes an inferred state in which none (or very few) of the cells actually exist!

Figure 1. Single cell RNA-seq reveals cellular heterogeneity that is masked by bulk RNA-seq methods.
Figure 1. Single cell RNA-seq reveals cellular heterogeneity that is masked by bulk RNA-seq methods.

Perhaps you are a neuroscientist, working in the mouse brain, studying the development of a subpopulation of neurons that produce a specific neurotransmitter. If only you could isolate that population and analyze it on a cell-by-cell basis, the potential insights would be, indeed, neuronally stimulating! Similarly, in the case of cancer research: how do you best tease apart gene expression and, perhaps more importantly, cellular heterogeneity in a tumor? What if you could compare not only inter-tumor, but intra-tumor differences and explore the immune cell populations related to the tumor microenvironment? This diversity, often obscured by bulk RNA-seq methods, can be revealed by using single cell RNA-seq to specifically explore these clinically significant cell populations. The examples of drawbacks to bulk analysis are abundant, and dissecting cell-type differences in most, if not all, of these complex biological systems is critical to our understanding of their contributions, especially during development and in the progression of disease.

How do we isolate and analyze single cells?

10x Genomics’ single cell RNA-seq (scRNA-seq) technology, the Chromium Single Cell 3’ solution, allows you to analyze transcriptomes on a cell-by-cell basis through the use of microfluidic partitioning to capture single cells and prepare barcoded, next-generation sequencing (NGS) cDNA libraries. Specifically, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil are combined on a microfluidic chip to form reaction vesicles called Gel Beads in emulsion, or GEMs. GEMs are formed in parallel within the microfluidic channels of the chip, allowing the user to process 100’s to 10,000’s of single cells in a single 7-minute Chromium instrument run. It’s important to note that cells are loaded at a limiting dilution in order to maximize the number of GEMs containing a single cell and ensure a low doublet rate, while maintaining a high cell recovery rate of up to ~65%.

Each functional GEM contains a single cell, a single Gel Bead, and RT reagents. Within each GEM reaction vesicle, a single cell is lysed, the Gel Bead is dissolved to free the identically barcoded RT oligonucleotides into solution, and reverse transcription of polyadenylated mRNA occurs. As a result, all cDNAs from a single cell will have the same barcode, allowing the sequencing reads to be mapped back to their original single cells of origin. The preparation of NGS libraries from these barcoded cDNAs is then carried out in a highly efficient bulk reaction. The video below provides a great visual explanation of how this all works.

From sample prep to how-to videos: Helpful links for getting started

The Chromium Single Cell 3’ workflow begins with your cells of interest, followed by NGS library construction using our reagent kits and the Chromium Controller. Preparing your barcoded cDNA library (as generally described in the above video) is an important step, and true to the saying "garbage in, garbage out"—preparing high-quality cell suspensions is key to getting the most out of your RNA-seq data. If you need assistance with the cell preparation process, we have multiple demonstrated protocols available on our website, such as dissociation of neural tissue or preparation of moss protoplast suspensions for use in scRNA-seq, with more protocols being added regularly to our support website. For guidance and recommendations on how to best prep your samples once they are in suspension, a good starting point is our single cell preparation guide, where you can learn more about best practices and view a general protocol. If you are more of a visual learner, our how-to videos should help walk you through the sample and library preparation process.

Analyzing your single cell data

After your library is prepared, sequencing is performed using Illumina® sequencing instruments. This leads to the fun part: gleaning biological insights from your single cell RNA-seq data! The (potentially) massive amount of data you generate from the Chromium Single Cell 3’ solution can be easily handled by our software and visualization tools, which were designed to take away a lot of the guesswork: Cell Ranger pipelines transform the barcoded sequencing data into files ready for single cell expression analysis, and visualization is accomplished via Loupe Browser. If you would like a step-by-step overview, these tutorials on the software support site cover everything from transcript counting with Cell Ranger to beautiful data visualization with Loupe Browser.

There are also more than a thousand tools developed by the community for single cell RNA-seq analysis. We put together some tips for continuing your journey after running Cell Ranger. Read more about data analysis topics on our Analysis Guides website, where we summarize important analysis topics, provide guides and tutorials, and share up-to-date info on all things 10x Genomics data analysis.

If you have any other questions or concerns about getting started with the Chromium Single Cell 3’ solution, feel free to contact us directly. Whether it be tracking brain organoid development, studying bone marrow transplantation in patients with acute myeloid leukemia, or unraveling a novel pathway for intestinal stem cell self-renewal, the number of Chromium Single Cell publications are growing daily, and we look forward to hearing about all of your amazing research!

10x Genomics, Chromium, Loupe Browser, Cell Ranger and certain other marks are trademarks and registered trademarks of 10x Genomics, Inc.

Editor’s Note: This post was originally published in 2017 and has been updated in accordance with the current 10x Genomics style guidelines, accurate resource links, and information about new additions to the Chromium Single Cell technology platform.