A Review of Droplet-Sequencing
Cells, the building blocks of tissues, organs, and organisms, vary broadly in type and state. Today, the lack of information about cellular diversity in complex tissues leads researchers to find new ways of single-cell analysis and characterization. This new type of analysis could be very useful for the detection of cancer, tumors. Almost anything that is likely to have diversity in the population of cells and would allow to make great progress in these fields.
The emergency of microfluidics brings a lot of new opportunities into the biomedical field. Drop-Seq presents a microfluidics-based method for quickly profiling thousands of individual cells simultaneously in parallel, inexpensive and easy experiments, using droplet generation. Each cell is compartmentalized in one droplet, where it is associated to a bar-coded microparticle and is subjected to several reactions. This process allows to analyze cell mRNA transcripts while remembering the transcripts’ cell of origin.
Drop-seq consists of the following steps:
The first step aims at dissociating a complex tissue into individual cells, in order to obtain a single-cell suspension.
The second step consists in preparing the bar-coded microparticles. Each primer is composed of three parts: a “PCR handle,” a “cell barcode” and a unique molecular identifier (UMI). PCR handle is the same for all primers and beads and allows to prime PCR amplification once STAMPs obtained. On each bead, cell barcodes are the same on all primers, but differ from the other beads, and allow to recover the cells’ origin. Finally, UMIs are different on each primer and avoid double counting transcripts. The barcode generation is driven by 12 “split and pool” cycles, where each time, one of the four DNA bases is added. Finally, to synthesize the UMIs, eight rounds of degenerate synthesis occur with the four DNA bases.
Thirdly, a microfluidics device is used to co-encapsulate each cell individually with a distinctly bar-coded microparticle, in a tiny droplet. The geometry of the chip allows to join two aqueous flows, one containing cells, the other containing bar-coded beads in a lysis buffer, before they are compartmentalized and mixed into discrete droplets.
Figure 1. Schematic of droplet formation and cell encapsulation in a microfluidic device
Figure 2. Example of a microfluidic device for Drop-Sequencing and flow delivery rates for oil, cells and beads
The next step aims at lysing the cells and hybridizing the RNA. Once isolated in droplets, cells are lysed and release their mRNAs, which then hybridize with the primers.
Once the reactions in the droplets completed, the latters are broken by adding a reagent which destabilizes the oil-water interface. All the microparticles are collected and washed, in order to generate thousands of “single-cell transcriptomes attached to microparticles” (STAMPs) at once by reverse-transcribing mRNAs into cDNAs.
In order to have plenty of individual cells to analyze, bar-coded STAMPs are then amplified by PCR reaction.
The final step consists in sequencing and analyzing the resulting molecules and to recover each transcript’s cell of origin. Reads are organized by their cell barcodes, and double counting is avoided thanks to UMIs. Finally, a matrix of digital gene-expression measurements can then be established for further analysis.
This concise review article was written by Wilfried Sire from Elveflow Microfluidics and been posted with permission from Elveflow. Readers can find out more about their work here. Other related information can be found through
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