scRNA-seq is a breakthrough technique that allows researchers to examine cell heterogeneity, identify rare cell populations and explore gene co-expression patterns. It has been crucial for expanding our knowledge of complex biological processes and developing new therapies in the fields of neurology, diabetes, cardiovascular disease and cancer – among many others – and has evolved rapidly in the short time since its first use in 2009.1 Third generation scRNA-seq incorporating combinatorial barcoding shows immense value for medical research, as it can be used to study cellular responses to drug treatments, understand the genetic mechanisms behind them, and determine the efficacy of stem cell therapies in different disease areas. It also offers unprecedented throughput, bypassing the need for physical cell partitioning – and associated expensive equipment – necessary for previous iterations of scRNA-seq.

Revolutionizing single cell sequencing 

Parse Biosciences, based in Seattle, Washington, USA, specializes in developing groundbreaking tools for third generation scRNA-seq at scale. Its proprietary Evercode™ combinatorial barcoding technology labels molecules with cell-specific combinations of barcodes, enabling projects to scale up without the need for cell isolation. The Evercode scRNA-seq workflow consists of 3 sections: combinatorial multi-step barcoding of single cells, amplifying barcoded cDNA and, finally, preparing libraries for sequencing. In the first section, cells are fixed and permeabilized, enabling each individual cell to become its own reaction chamber and removing the need to capture individual cells in droplets or microwells. The split-pool barcoding process then labels cells with a large number of barcode combinations.

Up to 1 million cells and 96 biological samples can be processed in parallel using Evercode technology. Cells can be collected, fixed and stored for up to 6 months, allowing samples collected on different days to be processed together, such as in time course studies or to avoid batch effects. The gene and transcript detection technology from Evercode has demonstrated superior performance to droplet-based methods, thanks to its high sensitivity at scale. The technique also yields high quality data, which can be analyzed and shared using Trailmaker™ Parse Biosciences’ end-to-end data analysis software, which is provided alongside the sequencing kit.

Dr Charlie Roco, co-founder and CTO of Parse Biosciences, highlighted the importance of automation for this advanced scRNA-seq technique: “The large-scale adoption of combinatorial barcoding for RNA sequencing has been limited for several reasons. For example, many sequencing and automation instruments currently on the market can be cost prohibitive for smaller facilities with tighter budgets, and can take months to set up and fully train staff to use them. The lack of user-friendly and straightforward platforms at a realistic price point is a roadblock in scRNA-seq adoption, highlighting the need for a more accessible solution to encourage wider uptake.”

Integrating automation for streamlined barcoding

Parse Biosciences reached out to INTEGRA in late 2023 to co-develop an automated scRNA-seq solution that would help remove some of the barriers hindering more rapid progress in the field. Through the successful collaboration with INTEGRA, the first of the 3 Evercode workflow phases has now been automated on the ASSIST PLUS pipetting robot, using the D-ONE single channel pipetting module and an 8 channel VIAFLO lightweight electronic pipette. Developing and verifying this trailblazing protocol took only a few months, and the automation bundle was launched at the start of May 2024. The ASSIST PLUS equipped with the D-ONE pipetting module adds well-specific barcodes to intracellular transcripts, prepares reagent mixtures, and dispenses cells into the Evercode kit’s barcode labeling plates, enabling single cell discrimination. This seamless integration helps to reduce human error and inter-operator variability, as well as allowing users to increase their lab’s throughput, easily sequencing hundreds of separate samples in a single week.

Charlie continued: “This easy-to-use, cost-effective, powerful liquid handling set-up has made automated sample preparation for scRNA-seq far more streamlined and accessible to labs of all sizes and budgets, including academic groups, genomics core facilities and pharmaceutical companies. Although it is still only early days, our customers have already fed back to us on how liquid handling automation has dramatically sped up their sequencing processes for faster throughput. In addition, it has helped them to standardize scRNA-seq by reducing errors, leading to more reproducible results. In fact, our data so far shows that labs have been able to reduce their hands-on time by up to 75 percent and experience up to an 85 percent reduction in manual pipetting steps, providing the added benefit of freeing up highly trained staff to perform other vital tasks and begin new scRNA-seq research projects.”

“We’re now in the process of automating the remaining 2 sections of our scRNA-seq protocol on the ASSIST PLUS, and anticipate making this updated solution available to customers in the near future. We’re very excited to continue our successful collaboration with INTEGRA, and look forward to contributing to this fast-moving field of research in the coming months and years,” Charlie concluded.

More information: Product sheet Parse and INTEGRA single cell automation bundle

Reference

1. Tang F, Barbacioru C, Wang Y, et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009;6(5):377-382. doi:10.1038/nmeth.1315