- Cancer research
- Career spotlights
- Current events
- Customer stories
- Gene editing
- Research news
- ABRF publishes single-cell RNA-seq benchmarking study
- Two RNA-seq approaches reveal resident memory T cells hold the power to reverse liver fibrosis
- Combination of single-cell RNA-seq approaches yields insights into the brain
- Combining droplet and full-length sequencing technologies for a complete picture
- Smashing single-cell sequencing sensitivity
- Change of heart: exploring transcriptional variation in cardiomyocytes
- Accelerating chromatin mapping with single-cell ATAC-seq
- Bringing epigenomic profiling to the single-cell biology stage
- Using the power of RNA-seq to characterize brain cell types
- Stem cell research
- Tips and troubleshooting
- Women in STEM
- That's Good Support!
- About our blog
Change of heart: exploring transcriptional variation in cardiomyocytes
We, as researchers, find ourselves fascinated by and drawn to particular biological phenomena. The research topics we choose commit us to working with specific cell types or biological models, sometimes with surprising limitations. One frustration that researchers might face is not being able to perform a desired experiment because their cell type is not compatible with the available methods.
To eliminate these cell-type based restrictions, we designed the ICELL8 system to be flexible and open so that researchers can enjoy the benefits of single-cell sequencing, even with cell types that have previously proven challenging for this method. In this installment of the ICELL8 Innovator Series, we shine the spotlight on researchers from the Max Planck Institute for Heart and Lung Research, who developed a method to perform scRNA-seq on intact adult mouse cardiomyocytes. (Access the step-by-step protocol for mouse cardiomyocyte preparation using the ICELL8 system.)
Matters of the heart
While most somatic cells are diploid, the number of chromosomes in adult cardiomyocytes can vary, with the most common arrangement being two nuclei with two chromosome sets per cell. Yekelchyk et al. wanted to examine the transcriptomes of mono- and multi-nucleated cardiomyocytes to determine if differences existed and whether those variations have functional consequences.
Single-cell RNA-sequencing is an obvious choice for answering this question, but the authors note that this method has not been previously applicable to intact adult cardiomyocytes. One key reason is that the very large size of the cardiomyocytes is not compatible with microfluidic- and droplet-based scRNA-seq methods. Another complication is that information about nuclei numbers is lost when performing single-nuclei sequencing unless stringent presorting based on nuclei numbers is performed.
Only the ICELL8 system enabled us to address similarities and differences associated with the nuclearity of cells. Moreover, large cells such as cardiomyocytes are easily damaged during isolation, causing profound changes in the RNA content. The ICELL8 system allows rigorous quality control of analyzed cells, which is very helpful to avoid misinterpretation of sequencing data."
—Dr. Thomas Braun, corresponding author
To overcome these challenges, the researchers turned to the ICELL8 system, which features a large-bore nozzle capable of accommodating the large size of the cardiomyocytes (up to 200 µm long). Since cardiomyocyte suspensions are prone to sedimentation over time, the researchers took advantage of the flexibility of the platform to modify the dispensing protocol to frequently mix the cell suspension, resulting in a more uniform distribution of the cardiomyocytes across the ICELL8 chip (Figure 1).
Don't miss a beat
A preliminary principal component analysis of the scRNA-seq data indicated that the cardiomyocytes formed two distinct clusters, but closer inspection revealed that the clustering seemed to be related to the absolute number of genes detected (Figure 2, Panel A). Because the ICELL8 instrument has an integrated imaging step following the cell dispense, the authors were able to view each cell and correlate them to the sequencing results. This detailed analysis revealed that the cells that formed Cluster 1 appeared to have experienced RNA degradation due to cellular damage as evidenced by the loss of the rod-shaped morphology, small size, and hazy cell definition (Figure 2, Panels A and B).
Again, the researchers took advantage of the openness of the ICELL8 platform to adjust the imaging-based cell selection criteria so that only those wells with intact, undamaged cardiomyocytes would be processed and analyzed. With an average of 0.6 million reads and 3.9 thousand genes detected per cell, the scRNA-seq data revealed a relatively homogeneous distribution of the cardiomyocytes despite differences in size, indicating that increased ploidy has minimal impact on the cardiomyocyte transcriptome (Figure 2, Panel C).
The researchers went on to investigate whether heterogeneity could be induced through pathological conditions such as cardiac hypertrophy. Following the induction of cardiac hypertrophy, they discovered transcriptional differences that seemed to be driven by differences in the local tissue environment rather than by the ploidy of the cardiomyocytes.
Making single-cell sequencing more accessible
Single-cell sequencing is an invaluable tool for understanding cellular behavior under wild-type and disease conditions. However, without the proper tools, the application of this technique has not been possible for many cell types. ICELL8 technology was designed to allow the dispensing and processing of a wide range of cell sizes, from small nuclei to large cardiomyocytes. Additionally, the system provides the flexibility to develop and optimize new assays for custom applications, plus the ability to tie your sequencing data back to the processed cells, giving researchers a deeper understanding of their data. This use of the ICELL8 system by the Max Planck researchers—bringing scRNA-seq to intact cardiomyocytes—is just one of many examples of how researchers are using this open, flexible system to achieve their scRNA-seq goals. There are more innovators to come, so fill out the form below to be the first to know when we release additional ICELL8 Innovators Series articles.
Yekelchyk, M., Guenther, S., Preussner, J. & Braun, T. Mono- and multi-nucleated ventricular cardiomyocytes constitute a transcriptionally homogenous cell population. Basic Res. Cardiol. 114, 36 (2019).
Back to Blog Front
Sign up to receive notifications for new ICELL8 Innovators Series posts.
Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2022 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.