Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger

Abstract

Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre-or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.

Dual‐layered hydrogels allow complete genome recovery with nucleic acid cytometry

“…[64][65][66] These attributes make PTE-NAC a powerful strategy for isolating specific cells, viruses, or DNA sequences of interest from heterogenous suspensions, opening up unique opportunities in the characterization of understudied microbes in ecological and microbiome samples, [3] isolation of genomic islands for cell engineering and genetic disorders, [17,25] and deep characterization of disease causing genomic alterations in human cells, including HIV and cancer. [17,18]…”

“…[ 11 ] Microfluidic droplet encapsulation with polymerase chain reaction (PCR) screening affords a unique and powerful approach for detecting specific cell types based on nucleic acid biomarkers and, when combined with active or inertial microfluidic sorting, affords superb enrichment of rare subpopulations. [ 16–22 ] This paradigm, called nucleic acid cytometry (NAC), [ 23 ] is capable of multiplexed DNA and RNA sequence detection, [ 18 ] can be integrated with conventional single‐cell antibody staining methods, [ 13,24 ] and can isolate cells, [ 18,25 ] viral particles, [ 26 ] and DNA strands [ 27,28 ] from complex mixtures. The application of NAC has enabled the isolation of numerous cell types, viruses, and nucleic acids that would otherwise have been impossible to capture.…”

Single-cell droplet microfluidics for biomedical applications

“…[116][117][118][119] Because of its high fluxes, controllable size, and use of independent components, microfluidic droplet technology has attracted a significant amount of attention as a tool for cell-based high-throughput analysis in biomedical applications such as proteolytic activity detection, 79,120,121 cytokine secretion, 76,122 and gene sequencing. 123 Here, we review single-cell droplet applications-including small-molecule detection, protein analysis, drug screening analysis, and genetic analysis-that have been introduced in biomedical fields over the last six years.…”

Simple One-Step and Rapid Patterning of PDMS Microfluidic Device Wettability for PDMS Shell Production

“…Droplet-based microfluidics have emerged as a promising tool due to its effective and exquisite control and operation ( Jeong et al, 2021 ). The water-in-oil (w/o) droplets generated by microfluidic devices, which can be used as microreactors, have been widely applied in various fields, including pharmaceutical ( Herranz-Blanco et al, 2014 ; Pessi et al, 2014 ; Kong et al, 2015 ), biochemical ( Chen et al, 2009 ; Seiffert et al, 2010 ; Thiele et al, 2010 ; Abate et al, 2011 ; Mary et al, 2011 ; Clark et al, 2020 ), physics ( Kanai et al, 2010 ; Choi et al, 2019 ; Shi et al, 2020 ), food ( Lu et al, 2016 ; Zhang et al, 2016 ), cosmetic ( Li et al, 2018 ; Sun et al, 2020 , 2021 ; Jeong et al, 2021 ), agriculture ( Chu et al, 2007 ; Neethirajan et al, 2011 ), and in genetic screening ( Macosko et al, 2015 ; Zhang et al, 2015 ; Wang et al, 2020 ). However, the process of microdroplet formation is often accompanied by satellite small droplets either generated in flow-focusing orifices or separated from target droplets by spontaneously emulsification due to mechanical breakup or chemical instability ( Schmitt et al, 2017 ; Zabar et al, 2020 ).…”