Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow.
Single-cell RT-LAMP mRNA detection by integrated droplet sorting and merging
Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies.
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- Award ID(s):
- 1708706
- NSF-PAR ID:
- 10108056
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 19
- Issue:
- 14
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 2425 to 2434
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Droplet microfluidics has become an indispensable tool for biomedical research and lab-on-a-chip applications owing to its unprecedented throughput, precision, and cost-effectiveness. Although droplets can be generated and screened in a high-throughput manner, the inability to label the inordinate amounts of droplets hinders identifying the individual droplets after generation. Herein, we demonstrate an acoustofluidic platform that enables on-demand, real-time dispensing, and deterministic coding of droplets based on their volumes. By dynamically splitting the aqueous flow using an oil jet triggered by focused traveling surface acoustic waves, a sequence of droplets with deterministic volumes can be continuously dispensed at a throughput of 100 Hz. These sequences encode barcoding information through the combination of various droplet lengths. As a proof-of-concept, we encoded droplet sequences into end-to-end packages ( e.g. , a series of 50 droplets), which consisted of an address barcode with binary volumetric combinations and a sample package with consistent volumes for hosting analytes. This acoustofluidics-based, deterministic droplet coding technique enables the tagging of droplets with high capacity and high error-tolerance, and can potentially benefit various applications involving single cell phenotyping and multiplexed screening.more » « less
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null (Ed.)Quantification of cell-secreted molecules, e.g. , cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.more » « less
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Abstract Mechanical properties have emerged as a significant label-free marker for characterizing deformable particles such as cells. Here, we demonstrated the first single-particle-resolved, cytometry-like deformability-activated sorting in the continuous flow on a microfluidic chip. Compared with existing deformability-based sorting techniques, the microfluidic device presented in this work measures the deformability and immediately sorts the particles one-by-one in real time. It integrates the transit-time-based deformability measurement and active hydrodynamic sorting onto a single chip. We identified the critical factors that affect the sorting dynamics by modeling and experimental approaches. We found that the device throughput is determined by the summation of the sensing, buffering, and sorting time. A total time of ~100 ms is used for analyzing and sorting a single particle, leading to a throughput of 600 particles/min. We synthesized poly(ethylene glycol) diacrylate (PEGDA) hydrogel beads as the deformability model for device validation and performance evaluation. A deformability-activated sorting purity of 88% and an average efficiency of 73% were achieved. We anticipate that the ability to actively measure and sort individual particles one-by-one in a continuous flow would find applications in cell-mechanotyping studies such as correlational studies of the cell mechanical phenotype and molecular mechanism.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C9LC00161A
