Microfluidics-free Method for Single-Cell Sequencing

[seqadmin]

A recent publication in Nature Biotechnology reveals a templated emulsification-based single-cell RNA sequencing technology that does not use microfluidics. This new method, particle-templated instant partition sequencing (PIP-seq), encapsulates and lyses single cells and then barcodes the cDNA in uniform droplet emulsions using only a vortexer.

Despite the array of commercially available single-cell sequencing methods, PIP-seq was developed because there were no existing single-cell methods that could accommodate a wide range of throughputs in addition to not requiring expensive equipment or customization. PIP-seq was designed to handle a variety of sample formats, from microwell plates to large conical tubes, allowing users to process thousands to millions of cells with ease.

The PIP-seq process begins with particle-templated emulsification, where single cells are mixed with lysis reagents and barcoded hydrogel templates in monodispersed water-in-oil droplets. After vortexing the mixture, the cells are encapsulated and then lysed by increasing the temperature to 65 °C. The increase in heat activates proteinase K, allowing the mRNA captured on polyacrylamide beads containing unique barcodes to be released. At this point, the procedure can be stopped, and samples can be stored at 0 °C for processing at a later date.

Once the process resumes, the oil is removed from the samples, and the polyacrylamide beads are transferred into a buffer for reverse transcription of the mRNA. The resulting full-length cDNA is amplified, tagmented, and converted into the appropriate sequencing libraries.

To validate the efficiency of PIP-seq, the researchers responsible for its development tested it on a variety of different samples. In mouse-human mixing tests, they were able to demonstrate high-purity transcriptomes with very low levels (below 3%) of cross-contamination. They were also able to use PIP-seq with multiomics measurements and to accurately characterize cell types in human breast tissue

In addition, PIP-seq was used for single-cell transcriptional profiling of mixed phenotype acute leukemia. The downstream analysis identified transcriptional heterogeneity that was hidden using immunophenotype observations alone. The researchers also detected a modulation of ribosomal genes that they believe could be linked to treatment resistance for this type of leukemia.

With all of these results taken together, the research team has demonstrated that PIP-seq can be used for scalable single-cell sequencing projects on a variety of applications. Commercially released prep kits utilizing this technology are currently only designed for Illumina sequencers, but users have reported successful results with Oxford Nanopore Technologies’ sequencers as well.

Recent developments with PIP-seq kits include a system for epitope sequencing that allows for multiplexed protein marker detection and cell hashing. It is the inventor’s goal to open up single-cell work to organizations without the funds or resources to pursue these types of experiments.