Journal: bioRxiv

Article Title: MusTer: A Generalizable Microfluidic Platform Combining Multi-Parametric Droplet Sorting and Multi-Droplet Merging in Single-Cell Sequencing

doi: 10.64898/2025.12.29.696950

Figure Lengend Snippet: Selected as a model, nuclei were extracted from Maize ( Zea mays B73 as a model) seedlings by the chopped leaves. Nuclei open chromatin DNA was fragmented by Tn5 transposase tagmentation and stained by SYBR Green I. Nuclei droplets were generated by co-encapsulating tagged nuclei and thermolabile proteinase K using the nuclei droplet generator . Multi-parametric singlet droplet sorting was performed on the droplet sorting chip embedded with a set of electrodes to identify and separate the singlet droplets from those empty and multiplet droplets. After sorting, proteinase K within the singlet droplets was activated at 37°C to denature Tn5 proteins on the open chromatin fragments. A collection of barcode droplets was separately generated through droplet digital PCR using the barcode droplet generator . The triplet-droplet merging module was designed to produce the PCR droplets encapsulating PCR reagents, and to match the generated PCR droplet with a reinjected sorted singlet droplet and a barcode droplet, forming a triple-droplet triad with the triple-droplet merging chip . The triple-droplet triad was merged by dielectrophoretic force on-chip. Droplet PCR was performed to amplify the tagged open chromatin DNA with specific barcode primer. Upon breaking the droplets, another round of bulk PCR was performed to introduce sequencing index i5, i7 and sequencing adapters. The produced library was purified for next generation sequencing. is created with BioRender.com.

Article Snippet: To optimize the accuracy in two-droplet and three-droplet pairing, as well as to reduce the probability of multiple nuclei singlet droplets pairing with barcode droplets at the same time, the flow rates were set as follows: Bio-Rad oil (for separating reinjected droplets) at 10 μL/min, PCR droplets generation Bio-Rad oil at 7.2 μL/min, reinjected nuclei singlet droplets at 2.35 μL/min, reinjected barcode droplets at 1.38 μL/min, and KAPA PCR reagent at 7 μL/min.

Techniques: Staining, SYBR Green Assay, Generated, Digital PCR, Introduce, Sequencing, Produced, Purification, Next-Generation Sequencing

Journal: bioRxiv

Article Title: MusTer: A Generalizable Microfluidic Platform Combining Multi-Parametric Droplet Sorting and Multi-Droplet Merging in Single-Cell Sequencing

doi: 10.64898/2025.12.29.696950

Figure Lengend Snippet: (a) qPCR results showing different amplification efficiency in Ct value from Tn5-tagged nuclei subjected to inactivation by SDS or proteinase K treatment compared to the control without Tn5 inactivation. (b) A representative microscopic image showing the barcode droplets, produced by ddPCR, where the fluorescently green droplets, stained by SYBR Green I, were considered positive, or those containing the barcodes. Quantitative analysis of the positive rate based on ∼3,000 droplets was then used to compute the barcode encapsulation rate shown in the bar graph, ∼18.9% and ∼2.5% containing single barcode template and multiple barcode templates, respectively. (c) Schematic of the triple-droplet merging chip layout, showing the labeled inlets for PCR reagents, oil, reinjected sorted nuclei droplet and as-prepared barcode droplets, as well as the outlet for merged droplets. Electrode channel was filled with 5 M NaCl solutions, and Ground channel was filled with 45 mM NaCl solutions. (d) Color-labeled microscopic images showing, (1) Region 1: On-chip generation of PCR droplet; (2) Region 2: Pairing of a PCR droplet and a reinjected singlet droplet; (3) Region 3: Matching of the PCR-singlet paired droplets and a barcode droplet; (4) Region 4: Merging of the triple-droplet triad. (e) The one-to-one pairing ratio of singlet droplets and PCR droplets, and matching rate of the triple-droplet triad. (f) Estimation of the nuclei utilization rate and data utilization rate with and without sorting. Error bars stand for mean ± standard variation with n = 3.

Article Snippet: To optimize the accuracy in two-droplet and three-droplet pairing, as well as to reduce the probability of multiple nuclei singlet droplets pairing with barcode droplets at the same time, the flow rates were set as follows: Bio-Rad oil (for separating reinjected droplets) at 10 μL/min, PCR droplets generation Bio-Rad oil at 7.2 μL/min, reinjected nuclei singlet droplets at 2.35 μL/min, reinjected barcode droplets at 1.38 μL/min, and KAPA PCR reagent at 7 μL/min.

Techniques: Amplification, Control, Produced, Staining, SYBR Green Assay, Encapsulation, Labeling

Journal: bioRxiv

Article Title: MusTer: A Generalizable Microfluidic Platform Combining Multi-Parametric Droplet Sorting and Multi-Droplet Merging in Single-Cell Sequencing

doi: 10.64898/2025.12.29.696950

Figure Lengend Snippet: (a) Schematic of the full chip layout, showing the labeled inlets for reinjected sorted singlet droplets, PCR reagents, as-prepared barcode droplets, salt solutions, and Bio-Rad oil, as well as the outlet for collecting the merged droplets. (b) Enlarged view of the merging region, where droplets are merged under an electric field. (c) Photograph of the actual fabricated triple-droplet merging chip.

Article Snippet: To optimize the accuracy in two-droplet and three-droplet pairing, as well as to reduce the probability of multiple nuclei singlet droplets pairing with barcode droplets at the same time, the flow rates were set as follows: Bio-Rad oil (for separating reinjected droplets) at 10 μL/min, PCR droplets generation Bio-Rad oil at 7.2 μL/min, reinjected nuclei singlet droplets at 2.35 μL/min, reinjected barcode droplets at 1.38 μL/min, and KAPA PCR reagent at 7 μL/min.

Techniques: Labeling