This article is based on a poster originally authored by Archana Bettadapur, Adam Barner, Melissa Netwal, Sanika Khare, Cam Jansen, and Dominic Skinner.
Many epigenetics applications leverage the power of single-cell DNA methylation sequencing, which provides unprecedented insight into cellular heterogeneity and gene regulation.
Scale Bio offers a user-friendly and fully kitted solution that makes single-cell methylation analysis accessible to more researchers. The company’s single-cell DNA methylation (scMET) kit features highly parallelized barcoding technology.
This article highlights the advantages of automating the Scale Bio single-cell methylation protocol, using SPT Labtech’s firefly® to enable streamlined analysis of single-cell methylomes isolated from human peripheral blood mononuclear cells (PBMCs). These PBMCs were compared against a library prepared by hand in parallel.
Introduction
To remove the need for complex instrumentation, Scale Bio utilizes the cell itself as a compartment to perform rounds of sequential barcoding in a plate-based workflow.
This technology has been successfully adapted to assess whole genome DNA methylation at the single-cell level, offering a high-throughput, affordable, and robust protocol able to improve diversity, yield, and coverage.
Figure 1. Scale Bio Single Cell Methylation Kit v1.1 Workflow. Bisulfite conversion and downstream library construction steps were automated on the SPT Labtech firefly. Image Credit: SPT Labtech
This approach considerably increases accessibility to single-cell analysis without requiring specialized instruments. However, implementing automation can significantly streamline processing time and reduce consumable consumption, while simultaneously reducing user-to-user variability and strain.
SPT Labtech’s firefly® liquid handling platform employs true positive displacement technology. The gentle approach of this technology makes it ideally suited to working with cell and nuclei suspensions.
Its ability to enable precise and dynamic dispensing with a notable reduction in dead volumes helps to reduce the plastic waste commonly associated with conventional pipetting methods.
Methods
Isolated PBMC nuclei were fixed, barcoded, sorted, and lysed according to the Scale Bio Single Cell Methylation Kit protocol. Lysed samples were stored at -20 °C for two months before being thawed on ice to enable downstream processing.
Reagents and master mixes were prepared off-deck according to the Scale Bio Single Cell Methylation Kit Protocol. They were then divided equally between the manual and automated libraries.
firefly® was used for the required chilled incubation, dispense, and vortex steps. Its plate and reservoir thermal modules were cooled to 0 °C to enable chilled processing.
Cell wash steps were performed manually using the ScaleBio spin funnel, which was also used to purify the final pooled indexed PCR library from a subset of wells.
The Agilent Tapestation DS5000 High Sensitivity DNA Kit was used to determine fragment size before sequencing on a NextSeq2000. Sequencing data from both the automated and manually generated libraries was analyzed with ScaleBio Seq Suite: MET v1.1.0.
Figure 2. Processing time by workflow step. The method developed on firefly reduced overall processing time by 35% and manual processing by over 50%. Image Credit: SPT Labtech
Figure 3. Reservoir and tip type comparison. Image Credit: SPT Labtech
*SPT Labtech firefly/dragonfly reservoir (1 g), SPT Labtech firefly/dragonfly syringe (4 g). †LP SBS 96-well diamond bottom reservoir weight (23.3 g) based on Axygen (RES-SW96-LP), other SBS reservoir types may differ. §Single tip weight (1.6 g) based non-filter 125 μL Apricot tips, other tip weights may differ. ‡Tip box not included in weight calculation.
Results
It was observed that both the manually prepared libraries and those generated via firefly® exhibited comparable metrics, maintaining robust cytosine coverage and high cell recovery.
Genome non-overlapping 5 Kb bins quantified by ALLCools (ALL methyl-Cytosine tools) performed cluster projection of the single nuclei.
Marker genes were identified using Differentially Methylated Regions (DMRs) and annotated based on known methylation signatures. This showcased the system’s high-resolution PBMC population imaging capacity based on known marker genes (Figure 3).
Figure 3. a) Library metrics of median unique reads, CG, and CH coverage were comparable across automated and manual libraries; b) and c) High median CG methylation and low median CH methylation demonstrates efficient bisulfite conversion in both library preparation methods. Image Credit: SPT Labtech
The UMAP cluster projection was colored by library type, confirming that the samples from both the manual and automated libraries were evenly distributed across clusters without bias from the library preparation method.
Figure 4. UMAP projection of the methylation data colored by cluster. Cell type annotation was performed using analysis of Differentially Methylated Regions (DMRs). Image Credit: SPT Labtech
Figure 5. UMAP projection of the methylation data clustered colored by library preparation method. Image Credit: SPT Labtech
Additional bioinformatic analysis highlighted an even representation of all cell types across both libraries, confirming that each preparation method offered comparable data quality and biological relevance (Figure 6).
Figure 6. Cell type proportions compared across automated and manually prepared libraries. Image Credit: SPT Labtech
Conclusion
The robust combination of Scale Bio’s easy-to-use, plate-based Single Cell Methylation Sequencing Kit and the efficient and sustainable platform afforded by the firefly® offers users an automated workflow able to lower workflow time by more than 35% and hands-on time by more than 50%.
Automating Scale Bio’s plate-based Single Cell Methylation Sequencing Kit on firefly® lowers workflow time by more than 35% and hands-on time by more than 50%.
The combination of these technologies enables the profiling of complex samples while maintaining specificity, sensitivity, and accuracy in identifying DNA methylation sites. It also enables detailed insight into cellular heterogeneity and trajectories, a key consideration when undertaking research into diseases, ensuring reproducible and consistent data sets as scientists scale and standardize their workflows.
Acknowledgments
Produced from materials originally authored by Archana Bettadapur, Melissa Netwal, Sanika Khare, Cam Jansen, and Dominic Skinner from Scale Biosciences; and Adam Barner from SPT Labtech.
About SPT Labtech
We Design and Manufacture Robust, Reliable and Easy-to-Use Solutions for Life Science
We enable life scientists through collaboration, deep application knowledge, and leading engineering to accelerate research and make a difference together. We offer a portfolio of products within sample management, liquid handling, and multiplexed detection that minimize assay volumes, reduce material handling costs and put the discovery tools back in the hands of the scientist.
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Many of our innovations have been born out of the desire to create solutions to existing customer problems; and it’s this ethos that drives SPT Labtech’s R&D efforts. Our strengths come from the trust our customers have with us to develop truly unique, automated technologies to meet their needs. We combine cutting edge science with first-rate engineering to put customers at the heart of everything we do.
A problem-solving state of mind
The substantial breadth of expertise within our company enables us to be involved in the full life cycle of our products from the initial design concept, mechanical and software engineering and prototyping, to final manufacture and sale. These qualities allow us to offer the best possible technical and mechanical support to all the equipment that we supply, hence maintaining excellent client relationships.
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