PIP-Seq- The Single Cell Experiment Without The Need Of Instrumentation

The booming single-cell technology is currently widely accepted by everyone. In addition to its widespread use in scientific research, single-cell technology is also widely used in antibody discovery, drug evaluation, targeted drug guidance and other fields.
At present, single-cell sequencing methods generally rely on microplate devices, microfluidic devices or fluid processing. Moreover, microfluidics, microplates and other devices are expensive, which has become a major barrier to single-cell technology research.

On March 6, 2023, Nature Biotechnology published an article about PIP-seq (Particle-templated instant partition sequencing) - Clark IC, Fontanez KM, Meltzer RH, et al. Microfluidics-free single-cell genomics with templated emulsification. Nat Biotechnol. 2023;41(11):1557-1566. doi:10.1038/s41587-023-01685-z. PIP-seq is a method that does not require specialized microfluidics equipment, expertise, or hardware. It is based on particle template emulsification, allowing people to encapsulate and barcode cDNA in a single cell in a uniform droplet emulsion using only a vortexer. Particle template instant partition sequencing (PIP-seq) is suitable for a variety of emulsion formats, including microplates and large-capacity conical tubes, and can process thousands of samples or millions of cells in a few minutes.

Figure: PIP-seq single-cell experimental flow chart
a, Schematic diagram of the emulsification process. Barcoded particle templates, cells, and lysis reagents are combined with oil and vortexed to generate monodisperse droplets.b. Cell lysis releases mRNA, which binds to beads coupled with barcodes and oligod(T).c. After de-oiling, reverse transcribe the mRNA into cDNA and perform the subsequent strand displacement step using TSO.d. Obtain full-length cDNA, which contains the barcode and the sequence required for Illumina sequencing.

Let's take a look at the data performance of PIP-seq in the literature published by the authors.
(A) PIP-seq twin rate performance
PIP-seq experiments were performed using mixed samples of humans and mice. The total number of cells captured was 1595, of which 12 were twin cells, with a twin rate of 0.75%. Below is the twin rate detection results.

(B) Single cell clustering
Using healthy breast samples, experiments were performed using both PIP-seq and 10x Genomics V3 reagents. The results showed that PIP-seq could distinguish luminal epithelial cells (LEP1 and LEP2), myoepithelial cells, fibroblasts, vascular cells, and two lineages of immune cells. The 10x data was extracted to reduce the number of captured cells to 2,400, and the sequencing volume of each cell was maintained at 36,500 reads. The final comparison showed that the PIP VS 10x Genomics results were 2,298 vs 1,757 unique genes, 7,491 vs 3,394 transcripts, and 2.34% vs 1.32% mitochondria. PIP-seq and 10x Genomics results were analyzed for marker genes, and the results showed that the marker gene analysis results were consistent.

(C) Other applications of PIP-seq
1. PIP-seq for CRISPR screening
PIP-seq was used to perform CROP-seq experiments, and the results showed that PIP-seq can be used for CRISPR screening. The transcriptome and gRNA sequencing using PIP-seq results was shown below:

2. Transcriptomic signature of MPAL recurrence

In Conclusion
The authors demonstrate that PIP-seq produces highly purified transcriptomes in mixed mouse-human studies, is compatible with multi-omics measurements, and can accurately characterize cell types in human breast tissue compared to commercial microfluidics platforms. Single-cell transcriptional profiling of mixed phenotype acute leukemias using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subpopulations that is hidden by standard immune phenotypes. PIP-seq is a simple, flexible, and scalable next-generation workflow that expands single-cell sequencing to new applications.

Product Recommendation
Hieff NGS® Single Cell/Low Input cDNA Synthesis & Amplification Module
Hieff NGS® Single Cell/Low Input RNA Library Prep Kit
Hieff NGS® C112P1 Fast Tagment DNA Library Prep Kit for Illumina® (for 1 ng)

References
Microfluidics-free single-cell genomics with templated emulsification by IC Clark, Nature Biotechnology, 41, pages1557–1566 (2023)