The single cell sequencing solution tries to unleash disruptive science with a vortexer

Fast and scalable template-based emulsification for single-cell genomics. a–d, PIP-seq enables encapsulation, lysis and barcoding of single cells. a, Schematic representation of the emulsification process. Barcoded particle templates, cells, and lysis reagents are combined with oil and vortexed to produce monodisperse droplets. b, Heat activation of PK leads to lysis and release of mRNA, which is captured on bead-bound barcoded poly(T)-oligonucleotides. c, Oil removal is followed by bulk reverse transcription of mRNA into cDNA. cTSO is the complement of the template switch oligonucleotide. d, Barcoded whole-transcriptome-amplified cDNA being prepared for Illumina sequencing. e.g. B. Efficient scale-up single bead-single droplet encapsulation. e, Particle template emulsification in tubes of different sizes (1.5 mL, 15 mL, and 50 mL) produces monodisperse emulsions capable of barcoding different cell numbers by orders of magnitude. f, PIP-seq is compatible with plate-based emulsification, including 96-, 384-, and 1,536-well plate formats. Representative images of experiments performed three times are shown. g, The estimated ability of different technologies to scale easily in terms of cell and sample numbers. Credit: natural biotechnology (2023). DOI: 10.1038/s41587-023-01685-z

A new single-cell cDNA encapsulation, lysis and barcoding method is faster and requires less equipment, hardware, cost and expertise. It has proven to be compatible and scalable with any size container, from 500ul microcentrifuge tubes to 50mL conical tubes, and also works with 96, 384 or 1,536 well microplates.

In their article “Microfluidics-free single-cell genomics with templated emulsification”, published in natural biotechnology, researchers estimate that 3,500 cells were barcoded with 35 µL of hydrogel template in a 500 µL tube, 225,000 cells with 2 mL of template in a 15 mL conical tube, and 1 million cells with 10 mL of template in a 50-tube tube can be -ml tubes. Regardless of tube size, only 2 min of vortexing is required for cell capture.

Particle-templated instant partition sequencing (PIP-seq) captures cells, barcoded templates, and lysis reagents into uniform, oil-coated water droplets with just a few minutes of vortexing. The method uses hydrogel particles forming >95% water solution as a template to obtain these well-defined droplets. The hydrogels with prefunctionalized beads are filled with single cells and heat-activated lysis solution during vortexing.

The cells are then lysed by raising the temperature to 65°C, which activates a proteolytic enzyme (proteinase K) that breaks down the cell plasma membrane and releases the cellular mRNA. The mRNA is then captured onto polyacrylamide beads decorated with barcoded sequences.

PIP-seq emulsions can be stored at 0°C with no change in data quality (72 hours), allowing multiple samples to be stacked for sequencing. After reconstitution, the oil is removed, the beads are transferred to reverse transcription buffer, and full-length cDNA is synthesized, amplified, and prepared for sequencing.

Single-cell transcriptomics is gaining increasing importance in research because it allows scientists to understand in minute detail what is going on inside a cell. Applications range from categorizing active cellular functions, determining disease etiology or progression, to uncovering hidden RNA regulators and specific interactions, many of which can only be roughly deduced in mass cell studies.

In the publication, the researchers demonstrate that PIP-seq produces highly pure transcriptomes in a mixed mouse-human cell assay. Look for the degree to which pre-lysed cells could lead to mRNA cross-contamination. The study found that the proportion of mouse reads in human transcriptomes was less than 3%.

PIP-seq was also tested in single cell transcriptional profiling of mixed phenotype acute leukemia. Here, the researchers identified transcriptional differences that go beyond observing immune phenotype. Modulation of ribosomal genes not previously associated with this type of leukemia could play a role in treatment resistance and suggest researchers a therapeutic target — an impressive find for a method-testing study that represents a blind spot to other methods.

Disruptive Innovation

When scientists speak of disruptive innovation or technology, they are generally referring to a new method that is changing the way research is done. Typically, this means making an existing process cheaper, faster, more accurate, more precise, or with an increased throughput of data collection. Much like cell phones replaced landlines, online streaming replaced video stores, or next-generation sequencing replaced Sanger sequencing, disruptive innovations are often adopted quickly, increasing the quality and speed of research work.

PIP-seq emulsion acquisition matches what can be achieved with microfluidic systems, but at a fraction of the cost and without major equipment investments, making it a simple, flexible, and scalable next-generation sequencing workflow that delivers the Expanding single-cell sequencing to new applications – making it a truly breakthrough innovation in single-cell research. The PIP-seq inventors hope this process will open up the field of single-cell sequencing for smaller, less-resourced research organizations and academic institutions to freely pursue the next wave of scientific discoveries.

More information:
Iain C. Clark et al, Microfluidics-free single-cell genomics with templated emulsification, natural biotechnology (2023). DOI: 10.1038/s41587-023-01685-z

Journal Information:
natural biotechnology

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