Capturing the Transcriptome and the Epigenome Simultaneously in Single Cells

In the last few decades, advances in analytical technologies have spurred scientists on a continual quest to gain deeper insights into cell biology, both in healthy and diseased states.

A toolbox of techniques now exists to analyze cells at the single-cell level, an approach which respects the heterogeneous nature of cells in tissues, organs and whole organisms. Single cell RNA sequencing, for example, enables the identification and quantification of mRNA present in a lone cell. Single cell epigenomic methods permit comprehensive profiling of the diverse epigenetic features of certain cells.

But how do we take our understanding of the different stages of the omics cascade to the next level? We analyze them simultaneously.

With the recent launch of its Chromium Single Cell Multiome ATAC + Gene Expression solution, 10x Genomics is making advances towards that goal. The new solution enables scientists to analyze both the transcriptome and the epigenome simultaneously, in the same single cell.

Technology Networks spoke with Ben Hindson, co-founder and chief science officer at 10x Genomics, to learn more about the capabilities of the solution, and how scientists are already using it.

Molly Campbell (MC): For our readers that may be unfamiliar, please can you distinguish between the epigenome and the transcriptome?

Ben Hindson (BH): The transcriptome is ultimately the by-product of a highly coordinated program of gene expression regulated by the epigenome. The epigenome is informed by your DNA’s chromatin state, which can be open (and accessible) or closed (and inaccessible). The 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.

MC: Why is it important to profile the epigenome and the transcriptome from the same cell? What challenges have prevented this from being possible before? 

BH: This multi-omic approach gives scientists the ability to link a cell’s epigenetic program to its transcriptional output, enabling a better understanding of cell functionality and bypassing the need to infer relationships through computer simulations.

Many previous studies have focused on single cell RNA-sequencing (RNA-seq), which is powerful for characterizing gene expression in different cell types. However, to understand what establishes gene expression differences it is important to profile the epigenetic program from the same cells.  Some researchers have generated separate single cell ATAC-seq and single cell RNA-seq datasets and tried to pair the data with computational tools to get both transcriptome and epigenome data. They then infer which cell types correspond between the two datasets.

Computational methods were an important advancement before the development of assays to directly measure RNA and ATAC in the same cell. Developing experimental methods have been challenging because they must capture two different analytes - mRNA mRNA and accessible DNA fragments – at the same time from the same cell, and ideally at the same sensitivity to the individual scRNA-seq assays. A lot of effort went into developing an assay that enables researchers to detect both chromatin accessibility and mRNA from nuclei with comparable sensitivity as each individual assay alone. The gene expression and chromatin accessibility data generated using the Chromium Single Cell Multiome ATAC + Gene Expression assay will enable researchers to probe their samples more deeply and gain a more holistic view of biology.     

MC: Please can you talk to us about how the Chromium Single Cell Multiome ATAC + Gene Expression solution was developed, and how it works?

BH: This solution is one of our most ambitious undertakings and it adds to the diversity of products that run on our Chromium platform for single cell analysis. It enables the insights scientists need in order to characterize how gene expression patterns are established and provides additional resolution to profile cell types and states using two modalities. Samples can be analyzed with greater depth and provide richer insights, including the discovery of new gene regulatory interactions or a better interpretation of epigenetic profiles with key gene expression markers.

With this solution, nuclei are first transposed using the enzyme transposase, which preferentially cuts nuclear DNA in open chromatin regions. Transposed nuclei are then partitioned into droplets, or GEMs, with a single gel bead that contains a unique 10x barcode. Within the GEM, the unique barcodes are attached to available mRNA and transposed DNA fragments in a single nucleus. Following this incubation, GEMs are broken and pooled before clean-up, pre-amplification and library construction. Two libraries are made from a single pool of GEMs, one for sequencing RNA and one for ATAC. RNA and ATAC fragments originating from the same nucleus are linked by their 10x barcode.

MC: Can you discuss any of the research that your customers are undertaking using this equipment? 

BH: Early customers already working with Chromium Single Cell Multiome ATAC + Gene Expression include Stanford University School of Medicine, Icahn School of Medicine at Mt. Sinai and Spain’s Centro Nacional de Análisis Genómico.

Dr Ansuman Satpathy, assistant professor of pathology at Stanford University School of Medicine is using this solution to understand the epigenetic and transcriptional regulation of immune cell dysfunction directly in patient samples to engineer more precise and effective immunotherapies.

Dr Holger Heyn, leader of the single cell genomics team at Spain’s Centro Nacional de Análisis Genómico, is working on delineating the dynamics underlying B-cell differentiation and activation. This multiome assay will enable his team to measure what before could only be predicted, enabling scientists to confirm how accurate prior predictions have been.

Dr Panagiotis Roussos, associate professor of genetics and genomics sciences, Icahn School of Medicine at Mount Sinai, is leveraging the technology to better understand the mechanisms affected by the non-coding risk genetic variation across a wide range of neuropsychiatric diseases, including Alzheimer’s, Parkinson’s, Schizophrenia bipolar disorder and major depression. 

MC: How do you think the technology will encourage advances in our understanding of human disease and personalized medicine?

BH: Scientists will be able to better establish the mechanisms of gene regulation in health and disease and also better understand the effects of genetic disruptions in non-coding regions. Genome sequencing studies have identified thousands of genetic variants associated with disease, but these often fall in non-coding regions of the genome and their function in disease is difficult to decipher. By enabling greater comprehension of cell functionality and the ability to accurately measure cellular relationships, this solution will contribute greatly to the extensive scientific knowledge base, ultimately resulting in new and more precise therapies for leading diseases.

Ben Hindson was speaking to Molly Campbell, Science Writer, Technology Networks.