Aug 24, 2017

Single-Cell RNA-Seq: An Introductory Overview and Tools for Getting Started

Sheila Clark

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

Complex biological systems result from the coordinated function 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 using 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 sub-population 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 to 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 gives 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 or your RNA-seq data. If you need assistance with the cell preparation process, we have multiple demonstrated protocols available on our website such as dissociating neural tissue or preparing moss protoplast suspensions for use in scRNA-seq, with more protocols being added regularly on 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, especially Chapters 4 through 7, should help walk you through the sample and library preparation process.

    Analyzing your data, one cell at a time

    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 that were designed to take away a lot of the guesswork—Cell Ranger™ Software transforms the barcoded sequencing data into files ready for single cell expression analysis, and visualization is accomplished via the Loupe™ Cell Browser. If you would like a step-by-step tutorial on using our software, Chapters 8 through 11 of our how-to-videos cover everything from transcript counting with Cell Ranger Software to beautiful data visualization with Loupe Cell Browser. There are also a growing number of single cell analysis tools developed by the community, including Seurat, Monocle and Cell View.  Read more about these analysis tools and other single-cell articles on the 10x Genomics Blog where we highlight important research contributions, provide tips and tricks, and keep you up-to-date on all things single cell!

    If you have any other questions or concerns about getting started with the Chromium Single Cell 3’ Solution, feel free to leave a comment here or 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!