Droplet-sequencing

Cells are the basic unit of biological structure and function. In most biological systems, our understanding of cell diversity is incomplete, such as the nervous system. The characterization of the identity and function of single cells, as well as the understanding of the functional capabilities and responses of each cell type, will accelerate biological discovery. However, today's technology does not yet provide a simple method to analyze a large number of individual cells simultaneously.

Drop-Seq is a strategy based on microfluidic technology, analyzing thousands of individual cells simultaneously by encapsulating cells in tiny droplets for parallel analysis. It uses a molecular barcode strategy to divide cells into nanoliter-sized reaction chambers through droplets to analyze their mRNA transcripts while remembering the source cell of the transcript. With this technology, researchers can simply conduct experiments in parallel and make 10000 single-cell libraries every day. Therefore, this method will allow the creation of a molecular profile of gene expression of known cell types and new candidate cell subtypes.

Microfluidic Drop-Seq

The advantages of Drop-seq benefiting from microfluidic technology include:

• High throughput results in a short time
• Reduce consumption of expensive samples
• Preparation of single-cell suspension from tissue
• Prepare barcode primer on the surface or inside of particles
• Use a microfluidic device to wrap each cell and particle together in a tiny droplet
• Once isolated in droplets, lyse the cells, which release their mRNAs, which then hybridize to the primers
• Break droplets and generate STAMP
• Amplify the STAMPs
• Sequencing and analysis-use STAMP barcodes to infer the source cell of each transcript

There are 2 types of microbeads that can be used in Drop-Seq: "simple" microparticles and hydrogel microparticles.

Figure 1. (a) Use a custom-designed microfluidic device to encapsulate individual cells together with microparticles/gel beads in droplets; (b) Two types of beads that can be used for Drop-Seq.

Each particle contains more than 108 individual primers, with the same "PCR handle" and "cell barcode", but with a different unique molecular identifier (UMI). The PCR handle is a constant sequence on all primers and beads, which allows PCR amplification after STAMP is formed. The cell barcode is only the same on all primers of the same particle, thereby restoring the source of the cells. Each primer has a different UMI, which can digitally count mRNA transcripts and identify PCR repeats. There is a 30 bp oligodT sequence at the end of all primer sequences, which is used to capture mRNA and initiate reverse transcription. A 30-bp oligodT sequence is present at the end of all primer sequences for capturing mRNAs and priming reverse transcription.

Once the single-cell suspension and microparticles are ready, custom-designed microfluidic devices can be used to encapsulate individual cells and microparticles in droplets.

Single-Cell RNAseq

The field of single-cell RNA sequencing has developed rapidly in the past few years, and it is now possible to analyze thousands of transcripts of each cell at the same time. The droplet-based scRNA-seq experiment is a complex multi-step process, but its general workflow will remain very similar and can be divided into 5 steps.

Figure 2. Schematic representation of a typical workflow for a scRNA-seq experiment. (Salomon R, et al. 2019)

Sample Preparation

Unless cell-cell interactions provide meaningful biological information, the sample must be broken down into a single-cell suspension, then use FACS, MACS, or other procedures to purify the sample to remove dead cells or enrich for cells of interest.

Wrapped in Droplets

After sample preparation, the cells are individually encapsulated in water-in-oil droplets. The core of all droplet-based scRNA-seq systems is the ability to create a single water-in-oil reaction chamber compatible with the cell scale. Researchers use droplet microfluidic systems (such as Drop-seq, inDrop, and Chromium 10X) to minimize costs while increasing cell throughput.

Barcode Incorporation and Molecular Amplification

Each cell contains a small amount of mRNA, so the transcription of each cell must be amplified for sequencing. The two methods used in the droplet-based scRNA-seq system are inherited from the molecular workflow of the previous scRNA-seq system and are based on in vitro transcription (IVT) or template switching amplification (TSO).

Sequencing

Currently, all common single-cell transcriptome platforms are designed around Illumina sequencing instruments and a technology called sequencing by synthesis (SBS). Regardless of the amplification method used, all materials are prepared by a molecular library, including the addition of adapters compatible with next-generation sequencing (NGS).

Data Processing and Informatics

The large amounts of data generated by NGS must be processed. More and more bioinformatics analysis frameworks have been developed to specifically manage, interrogate and interpret scRNA-seq data.

References

• Salomon R, et al. (2019). "Droplet-based Single Cell RNAseq Tools: A Practical Guide." Lab Chip. 19: 1706-1727.
• Moon H. S, et al. (2018). "Inertial-ordering-assisted Droplet Microfluidics for High-throughput Single-cell RNA-sequencing." Lab Chip. 18: 775-784.

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