Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing

Abstract

Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl, which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

Microfluidic Passive Valve with Ultra-Low Threshold Pressure for High-Throughput Liquid Delivery

“…As the processing rate of cell separation and concentration can be several mL/min, a valve with the function of high throughput flow control will be very effective for the miniaturization of the microfluidic cell sorting system [36,37]. In addition, the valve structure can be further optimized to reduce the flow rate for more available microfluidic applications, such as sample mixing [41], droplet manipulation [42], drug delivery [43], etc.…”

An Introduction to the Analysis of Single-Cell RNA-Sequencing Data

“…Droplet-based single-cell gene expression approaches, including DropSeq 13 and the commercial 10X platform, 14 , 15 , 16 , 17 , 18 , 19 use microfluidic chips to isolate single cells along with single beads in oil-encapsulated droplets, using microfluidics to bring oil, beads, and cell suspensions together in such a way that each droplet contains at most a single cell. 20 The beads are coated with DNA oligos that are composed of a poly(T) tail at the 3′ end for the capture of cellular mRNAs, and at the 5′ end both a cell barcode that is identical for every oligo coating an individual bead and a library of individual unique molecular identifier (UMI) barcodes of high diversity, each UMI different for every oligo on the bead ( Figure 1 A). 13 , 21 , 22 The transcripts from each individual cell captured and labeled by the DNA oligos attached to a bead within the droplets are reverse transcribed, amplified with PCR, and sequenced using a high-throughput platform, after breaking and pooling droplet contents.…”

Dean flow assisted single cell and bead encapsulation for high performance single cell expression profiling

“…In addition, particles were also subject to lift force, which was the net force of the inertial lift, caused by the shear gradient, and the wall lift, caused by the channel wall [21][22] . The balance of drag force and lift force dictated the equilibrium positions of the particles on the channel cross section 14 . Inertial microfluidics predicted that particles are more likely to occupy a single equilibrium position in a curvilinear channel when [21][22][23][24][25] designs of spiral channels would be sufficient to focus beads, but for cells, we added serpentine channels 15 at the outlet of the spiral channel as an additional focusing mechanism, as shown in Figure 1a.…”

“…In curved channels, the inertia of the fluids generates secondary swirling flow, named Dean flow, which accelerates the lateral displacement of the particles and thus facilitates the focusing. Based on the principle of inertial focusing, spiral channels were adopted in a reported work to order barcoded beads before droplet encapsulation 14 . Higher capturing efficiency and higher fraction of single bead encapsulation were reported.…”