Fast temporal single-cell measurement microfluidic devices

Abstract

Disclosed are microfluidic devices, systems, and methods for fast, temporal single cell measurements, referred to as "ChronoSeq." The disclosed ChronoSeq technology is able to analyze biological samples down to the single cell size for transient epigenomic changes in gene expression using single-cell RNA sequencing. In some aspects, a method for single-cell temporal measurements includes sequentially introducing cells and time-tagged microparticles into a microfluidic chip to co-encapsulate the cells and the time-tagged microparticles at a plurality of time points, wherein the time-tagged microparticles introduced at each time point include a unique temporal barcode; and generating droplets containing the co-encapsulated cells and time-tagged microparticles at each of the plurality of time points.

Description

PCT Patent Application Docket No. 009062.8580.WD00FAST TEMPORAL SINGLE-CELL MEASUREMENT MICROFLUIDIC DEVICES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent document claims priority to and benefits of U.S. Provisional Patent Application No. 63 / 755,877 titled “FAST TEMPORAL SINGLE-CELL MEASUREMENT MICROFLUIDIC DEVICES” filed on February 7, 2025. The entire content of the aforementioned patent application is incorporated by reference as part of the disclosure of this patent document.STATEMENT REGRADING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under contract AH 50282 awarded by the National Institutes of Health. The Government has certain rights in the invention.TECHNICAL FIELD

[0003] This patent document relates to biological sensors and analytical devices.BACKGROUND

[0004] A biological sensor or biosensor is an analytical tool that can detect a chemical, substance, or organism using a biologically sensitive component coupled with a transducing element to convert a detection event into a signal for processing and / or display. Biosensors can use biological materials as the biologically sensitive component, e.g., such as biomolecules including enzymes, antibodies, aptamers, peptides, nucleic acids, etc., or small molecules such as carbohydrates, as well as virus and living cells. Biosensors can use the transducer element to transform a signal resulting from the detection of an analyte by the biologically sensitive component into a different signal that can be addressed by a suitable transduction mechanism, for example, electrical, magnetic, mechanical, physicochemical, electrochemical, optical, piezoelectric, or others.SUMMARY

[0005] Disclosed are microfluidic devices, systems, and methods for fast, temporal single cell measurements, referred to as “ChronoSeq.” The disclosed ChronoSeq technology is able to analyze biological samples down to the single cell size for transient epigenomic changes in genePCT Patent Application Docket No. 009062.8580.WQ00expression using single-cell ribonucleic acid (RNA) sequencing.

[0006] In some embodiments in accordance with the present technology, a method for singlecell temporal measurements includes sequentially introducing cells and time-tagged microparticles into a microfluidic chip to co-encapsulate the cells and the time-tagged microparticles at a plurality of time points, wherein the time-tagged microparticles introduced at each time point include a unique temporal barcode; and generating droplets containing the co-encapsulated cells and time-tagged microparticles at each of the plurality of time points.

[0007] In some embodiments in accordance with the present technology, a method for temporal single-cell genomic sequencing includes providing a microfluidic chip having a cell inlet, a bead inlet, and an outlet; injecting a first sample of cells from a cell reservoir into the cell inlet of the microfluidic chip; injecting a first set of time-tagged beads from a first bead reservoir of a plurality of bead reservoirs into the bead inlet of the microfluidic chip, wherein the first set of time-tagged beads comprises a first time barcode; co-encapsulating cells from the first sample of cells with time-tagged beads from the first set of time-tagged beads in droplets within the microfluidic chip to produce a first time-tagged sample; collecting the first time-tagged sample from the outlet of the microfluidic chip; injecting a second sample of cells from the cell reservoir into the cell inlet of the microfluidic chip; injecting a second set of time-tagged beads from a second bead reservoir of the plurality of bead reservoirs into the bead inlet of the microfluidic chip, wherein the second set of time-tagged beads comprises a second time barcode different from the first time barcode; co-encapsulating cells from the second sample of cells with time-tagged beads from the second set of time-tagged beads in droplets within the microfluidic chip to produce a second time-tagged sample; and collecting the second time-tagged sample from the outlet of the microfluidic chip.

[0008] In some embodiments in accordance with the present technology, a microfluidic chip for temporal single-cell genomic sequencing includes: a substrate having a plurality of flow channels including a cell flow channel, a microparticle flow channel, and a dispersion fluid flow channel; a cell inlet coupled to the cell flow channel configured to receive cells; a microparticle inlet coupled to the microparticle flow channel configured to receive time-tagged microparticles; a dispersion fluid inlet coupled to the dispersion fluid flow channel configured to receive at least one of oil or air; a flow-focusing channel junction in fluid communication with at least one region of the cell flow channel, the microparticle flow channel, and the dispersion fluid flow channel,PCT Patent Application Docket No. 009062.8580.WO00wherein the flow-focusing junction is configured to receive a first fluid medium containing the cells, a second fluid medium containing the time-tagged microparticles, and the at least one of oil or air and to co-encapsulate cells with time-tagged microparticles in droplets, wherein the microparticle flow channel is configured as an inertial ordering channel comprising a spiral design to provide deterministic ordering of the time-tagged microparticles into the flow-focusing channel junction; and an outlet configured to output droplets containing co-encapsulated cells and time-tagged microparticles.

[0009] In some embodiments in accordance with the present technology, a system for temporal single-cell genomic sequencing includes: a microfluidic chip comprising a substrate having a plurality of flow channels, a cell inlet, a microparticle inlet, a dispersion fluid inlet, a flow-focusing junction, and an outlet, wherein the plurality of flow channels comprises a cell flow channel coupled to the cell inlet, a microparticle flow channel coupled to the microparticle inlet, and a dispersion fluid flow channel coupled to dispersion fluid inlet; a cell reservoir in fluid communication with the cell inlet of the microfluidic chip and configured to contain a plurality of cells; a plurality of microparticle reservoirs, each microparticle reservoir configured to contain time-tagged microparticles having a unique time-tag barcode, wherein each of the microparticle reservoirs is in fluid communication with the microparticle inlet of the microfluidic chip; a plurality of timepoint valves, each timepoint valve associated with a respective microparticle reservoir and configured to control injection of time-tagged microparticles from the respective microparticle reservoir into the microfluidic chip; and a controller comprising a processor and a memory, the controller configured to coordinate sequential opening of the plurality of timepoint valves to enable temporal barcoding of cells at different time points through co-encapsulation of the cells and selected time-tagged microparticles in the flow-focusing junction of the microfluidic chip.

[0010] The subject matter described in this patent document can be implemented in specific ways that provide one or more of the following features.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows a diagram illustrating an example embodiment of a ChronoSeq microfluidics device, in accordance with the present technology.

[0012] FIGS. 2A-2D show schematics illustrating temporal barcoding using an examplePCT Patent Application Docket No. 009062.8580.WO00embodiment of the ChronoSeq microfluidics device, in accordance with the present technology.

[0013] FIG. 3 shows a diagram depicting an example embodiment of a ChronoSeq Library Preparation workflow.

[0014] FIG. 4A shows a schematic depicting an example embodiment of ChronoSeq system including an example embodiment of a ChronoSeq microfluidics device in accordance with the present technology.

[0015] FIG. 4B shows a diagram of an example embodiment of a control device, in accordance with example embodiments of the ChronoSeq system shown in FIG. 4A.

[0016] FIG. 5 shows a diagram depicting an example implementation comparing an example ChronoSeq device and a conventional device.

[0017] FIGS. 6A and 6B show diagrams depicting an example embodiment and implementation of a ChronoSeq microfluidic chip, in accordance with the present technology.

[0018] FIG. 7 shows a diagram depicting an example bead modification workflow for creating ChronoSeq Time-Tagged beads.

[0019] FIG. 8 shows data plots depicting example results of a bulk RNA-Seq validation implementation for twelve Time-Tags.

[0020] FIG. 9 shows data plots depicting example results of a single-cell experiment to verify quality of data generated using the Single-Cell RNA-Seq Version of the disclosed technology.

[0021] FIG. 10 shows data plots depicting example results of example valuation implementations for bulk RNA-seq from K562 cells simulated with TNFa showing key timedelays and kinetic differences for gene activation.DETAILED DESCRIPTION

[0022] Single-cell technologies, like single-cell ribonucleic acid (RNA) sequencing (scRNA-seq), have revolutionized biological and medical research by offering detailed insights into cellular heterogeneity and function. As gene expression is inherently dynamic and versatile, it is essential to monitor temporal changes in response to perturbations or environmental cues to uncover regulatory mechanisms. While spatial transcriptomics and other omics technologies are advancing rapidly, there is a notable gap in high-resolution temporal omics techniques. Addressing this gap will enhance our ability to capture the full complexity of cellular processes, infer the regulatory causality in the genetic circuits, improve our understanding of disease heterogeneity, and uncoverPCT Patent Application Docket No. 009062.8580.WQ00mechanisms of drug resistance.

[0023] Current scRNA-seq technologies typically achieve a temporal resolution around 30 minutes at best. However, rapid transcriptional activity and gene expression changes can occur within minutes, making it difficult to infer the sequence of these changes and causal regulatory mechanisms underlying the cascades at the current temporal resolution. For example, NF-KB (nuclear factor-kappa B) responds extremely quickly to TNF-ot (tumor necrosis factor-alpha) stimulation. Notably, within 5 minutes of TNF-a exposure, a strong initial nuclear translocation of NF-KB occurs in cells. Also, for example, the nuclear NF-KB concentration can reach its peak around 25 minutes after stimulation and can induce early response genes, e.g., including cytokines and chemokines such as IL-6 (interleukin-6) and IL-8 (interleukin-6), as well as regulatory components of the NF-KB pathway like TNFAIP3 / A20 (tumor necrosis factor-alpha-induced protein 3, also known as A20). Thus, a half hour or more sampling using current scRNA-seq technologies would completely miss the initial activation and peak, which occur within the first 30 minutes. Moreover, this coarse temporal resolution by conventional approaches makes it challenging to infer the precise sequence of transcriptional cascades in the subsequent time periods.

[0024] Achieving minutes-level resolution for the current technologies often requires metabolic labeling, which is limited by cumbersome process(es) requiring separate labeling and library preparation at each timepoint, which in turn increases the possibility for reduced cell viability and potential perturbation. As an example, single-cell Global Run-On sequencing (scGRO-seq) is a technique to detect nascent RNAs and provides insights into transcriptional dynamics at minute scale. However, this method is limited by the number of cells that can be processed and the overall abundance of nascent RNA per cell, which is considerably lower than that of mature mRNA. It also has reduced efficiency in capturing promoter-proximal paused RNA polymerases, lower read depth compared to bulk methods, and technical complexity of using click chemistry.

[0025] Live-seq is an existing single-cell transcriptome profiling technique that enables RNA sequencing of individual cells without killing them. Live-seq samples RNAs from live cells but it involves sophisticated methods such as fluidic force microscopy (FluidFM) for non-destructive RNA collection, which may require specialized expertise and equipment. Moreover, the minimum time resolution is about 1 hour.PCT Patent Application Docket No. 009062.8580.WD00

[0026] Drop-seq is a low-cost, high-throughput single-cell RNA sequencing method that analyzes gene expression in thousands of individual cells simultaneously. It uses microfluidics to encapsulate single cells and barcoded beads into nanoliter- sized droplets, where mRNA is captured and labeled for identification, allowing the study of cell-to-cell heterogeneity. However, conventional Drop-seq methods and system lack the capability to temporally resolve samples, requiring manual loading of samples and involving specialized microfluidic setups that are subject to considerable maintenance costs, e.g., cleaning and unclogging. Yet, importantly, Drop-seq requires a substantial workflow time that risks cell viability, particularly for sequencing applications lasting over an hour to two hours. For instance, a temporal study using the conventional Drop-seq system requires each timepoint to be processed independently, which leads to a likelihood of inconsistent sampling or labelling times for each time point. For example, in each Drop-seq sample the metabolites used for labelling need to be washed out, which is not instantaneous, and the cells have time to incorporate more metabolites during processing delays; thereby increasing the chance of introducing an error of several minutes or longer for each sample (which in turn introduces the high possibility of measuring spurious perturbations, especially if the sampling rate is also several minutes).

[0027] Live-cell imaging is the only single cell technology that can observe temporal dynamics of gene expression in real-time. For instance, live-cell imaging has shown that single cells can exhibit oscillating activation of various transcription factors (TFs), such as NF-KB in response to TNF-a and p53 in response to deoxyribose nucleic acid (DNA) damage in individual cells. Importantly, this pulsatile behavior has a functional role, as blocking this pulsatile activity of p53 prevents cell recovery after DNA damage. However, live-cell imaging requires insertion of reporter genes in cells and can only monitor several genes, thus unsuitable for transcriptomic and direct analysis of, such as, drug response in patient samples. Live-cell imaging approaches are constrained by phototoxicity, weak signals, and limited genomic coverage, typically allowing visualization of only a few loci simultaneously. Fixed-cell methods like ATAC-PALM offer high spatial resolution but provide only static snapshots.

[0028] To address these and other problems, challenges, limitations, and issues with conventional single cell technologies, as well as add additional functionality, we developed a new droplet-based technology, which is referred to herein as “ChronoSeq.” ChronoSeq is designed for temporal single cell RNA-seq. The disclosed ChronoSeq technology offers unprecedentedPCT Patent Application Docket No. 009062.8580.WQ00temporal resolution, e.g., capturing data in short intervals, such as 7-minute intervals. ChronoSeq is configured to have an automated workflow that enables the preparation of all timepoints into a single cDNA library, significantly enhancing efficiency.

[0029] The disclosed ChronoSeq technology includes microfluidic devices, systems, and methods for fast, temporal single cell measurements. The disclosed ChronoSeq technology is able to analyze biological samples down to the single cell size for transient epigenomic changes in gene expression using single-cell RNA sequencing. For example, through time-barcoding and automated sampling, the disclosed ChronoSeq devices, systems, and methods add the missing time dimension to genomics. The disclosed ChronoSeq technology thereby creates a new paradigm by enabling temporal genomic characterizations, which provides substantial advantages over existing and conventional single cell technologies.

[0030] In some implementations, an example ChronoSeq device in accordance with the present technology disperses cell samples into nanodroplets which can be processed at higher throughput for lower costs than other array-based devices. Yet, unlike other conventional systems, the ChronoSeq device makes use of its additional features to improve the processing time of biological samples. This includes the addition of time-tags to microparticles (e.g., such as microscale- sized beads barcoded with nucleic acids) that gives a unique time stamp to different droplets collected during an analysis run such that a single run can measure RNA profiles across multiple time points from a single sample of cells (e.g., suspended cells) exposed to a test condition. Modifications to the cell suspension reservoir allow for greater durations of sample runs, and new washing cycles prevent sample cross-contamination. Also, some embodiments of the disclosed microfluidic device includes additional valves to control injection of desired time-tagged beads from different reservoirs, and they optimized the protocol to generate high-quality temporal data.

[0031] Example Embodiments of the Disclosed ChronoSeq Technology

[0032] Some embodiments of the disclosed ChronoSeq technology include a microfluidic device and / or system for analyzing biological samples down to the single cell size for transient epigenomic changes in gene expression using single-cell RNA sequencing, in short time intervals, e.g., including 7 minutes or less per interval. For instance, time intervals can be up to one minute or even 30 seconds, based on the amount of time-tagged microparticles utilized in the sample.

[0033] FIG. 1 shows a diagram illustrating an example embodiment of a ChronoSeqPCT Patent Application Docket No. 009062.8580.WQ00microfluidic device 100, in accordance with the present technology, for fast temporal single-cell measurements. The ChronoSeq microfluidic device 100 can be configured to sequentially coinject time-tagged beads and cells into a droplet-generating microfluidic chip at regular time intervals. As shown in FIG. 1, the ChronoSeq microfluidic device 100 includes a microfluidic chip 110, a cell reservoir 120 containing cells (e.g., suspended cells), and a plurality of bead reservoirs 130 each containing beads with a unique time-tag. The microfluidic chip 110 is in fluidic communication with each reservoir of the bead reservoirs 130 and is in fluidic communication with the cell reservoir 120. The bead reservoirs 130 are connected to corresponding timepoint valves 140 that control the injection of time-tagged beads into the microfluidic chip 110, e.g., via bead inlet 113 of the microfluidic chip 110. For example, the timepoint valves 140 facilitate the selection of the appropriate time-tagged beads for injection into the microfluidic chip 110. The cell reservoir 120 is configured to hold a cell suspension for injection into the microfluidic chip 110, e.g., via cell inlet 111 of the microfluidic chip 110. In some embodiments, the cell reservoir 120 (optionally) may include a magnetic stirrer assembly for cell suspension in the fluid medium contained in the reservoir, e.g., to maintain the cells evenly suspended. The cell reservoir 120 is connected to a cell sample valve 150 that controls the injection of the cells into the microfluidic chip 110. For each timepoint, for example, the cell sample valve 150 remains open while the corresponding timepoint valve of the plurality of timepoint valves 140 is opened. This allows for the co-injection of time-specific beads with cells, resulting in libraries barcoded with distinct time-tags. In some embodiments, the cell sample valve 150 includes one or more cell sample valves each coupled to a corresponding one or more cell channels to controllably provide the cells from the cell reservoir to the microfluidic chip 110.

[0034] In some embodiments, for example, the microfluidic chip 110 includes a flow-focusing junction, which is a region of the microfluidic chip 110 where the cells and time-tagged beads meet to facilitate co-encapsulation of the cells with time-tagged beads. For example, the flowfocusing junction can be configured at or near a central portion of the chip, which can optimize space allocation that can minimize the size requirements of the microfluidic chip 110 while effectively co-encapsulating cells with time-tagged beads.

[0035] In some embodiments, for example, the microfluidic chip 110 includes a third inlet 115 to controllably provide a dispersion fluid in the flow-focusing junction to intermix with the fluid mediums of the cells and the time-tagged beads. In various embodiments, the dispersion fluid canPCT Patent Application Docket No. 009062.8580.WQ00include an oil, air, or other medium to promote flow dynamics to facilitate interaction of the cells (e.g., nucleic acid constituents of lysed cells) with the time-tagged beads. As shown in FIG. 1, the third inlet 115 of the microfluidic chip 110 includes an oil inlet to controllably provide oil into the formation of the time-tagged cell-bead droplet at the flow-focusing junction of the microfluidic chip 110. It is understood that in some embodiments of the ChronoSeq microfluidic device 100, the third inlet 115 can be configured to alternatively inject air as the dispersion fluid. And, it is understood that in some embodiments of the ChronoSeq microfluidic device 100, the third inlet 115 can be configured as multiple inlets to inject multiple dispersion fluids, e.g., such as oil and air and / or another dispersive fluid with respect to the cell media and time-tagged beads media.

[0036] In some embodiments, for example, the ChronoSeq microfluidic device 100 includes an automated vortex mixer (not shown in FIG. 1, and shown later in FIG. 4A), e.g., which may be incorporated with the bead reservoirs 130, to keep the beads evenly suspended before injection. In some embodiments, for example, the the ChronoSeq microfluidic device 100 includes a magnetic stirrer assembly (not shown in FIG. 1), which can include a magnet positioned in each of the containers of the bead reservoirs 130 that contains the time-tagged beads, which are positioned over a magnetically-controlled stage to promote the rotation of the magnet to stir the time-tagged beads in their respective fluid media.

[0037] In various implementations, for example, the ChronoSeq microfluidic device 100 is included in a ChronoSeq microfluidic system that includes a control device (not shown in FIG. 1, and shown later in FIG. 4A), such as a computer or mobile communication device (e.g., smartphone, tablet, smartwearable, etc.). The ChronoSeq microfluidic device 100 can be controlled via serial connections to the control device, which coordinates the actions of its various components of the device 100. For example, the control device can be configured to operate the ChronoSeq microfluidic device 100 in two main phases during each injection cycle. For instance, in Phase 1, i.e.. a flushing phase, the fluid lines of residual beads or cells are cleared from previous injections; and in Phase 2, i.e. an injection phase, the ChronoSeq microfluidic device 100 precisely introduces cells and time-tagged beads into the microfluidic chip 110. This integrated device and system enables controlled, sequential introduction of cells and time-tagged beads, capturing gene expression dynamics across multiple time points in a single experiment.

[0038] In some embodiments, the ChronoSeq microfluidic device 100 can optionally include a cell culture system to maintain cell viability. As an example, depicted in FIG. 1, the ChronoSeqPCT Patent Application Docket No. 009062.8580.WQ00microfluidic device 100 can include a water bath to maintain the temperature of the cells in the cell reservoir 120 at 37°C and an air / pressure regulator to maintain gas flow and pressure (e.g., a 5% CO2 via a CO2 gas cylinder) that aids in cell culture. For example, in some embodiments, the device 100 includes a compressed air pressure controller in communication with flow sensors (not shown in FIG. 1, and depicted later in FIG. 4A) for each of the bead channels, cell channel(s), and oil channel(s), where the exemplary compressed air pressure controller enables rapid changes in flow rate, while the flow sensors provide the precise control needed for high-quality droplet formation during low flow rate operation.

[0039] In some embodiments, the ChronoSeq microfluidic device 100 is configured to support twelve unique time-tags with 7-minute intervals and 1-minute sampling duration, discussed in more detail later in this patent document.

[0040] Example Workflow of ChronoSeq Technology

[0041] In various implementations, the ChronoSeq microfluidic device 100 is configured to implement droplet-based single-cell characterization methods over array-based methods due to advantages of higher throughput and lower cost. In some implementations, for example, the ChronoSeq microfluidic device 100 can inject DNA barcoded beads, a single cell suspension, and a droplet generating oil into the exemplary flow-focusing junction at the same time. A barcode on is a unique, short sequence nucleotide attached to the microparticle (e.g., bead) that can track a parameter of a specific molecular measurement. In the ChronoSeq technology, the engineered barcodes are time-point trackers for the desired measurement. For instance, the ChronoSeq microfluidic device 100 incorporates an additional time-tag into DNA-barcoded beads that contain a cell barcode and unique molecular identifier (UMI), engineers a microfluidic chip-based device with additional valves to control injection of desired time-tagged beads from different reservoirs, and optimizes a temporal-sensitive protocol to generate high-quality temporal data.

[0042] FIGS. 2A-2D show a series of diagrams illustrating sequential timepoint injections using an example embodiment of the ChronoSeq microfluidic device 100 in accordance with some implementations of the disclosed technology. Each diagram in FIGS. 2A-2D depicts the state of the cell sample valve 150 and the timepoint valves 140 during a respective injection cycle.

[0043] As illustrated, the example ChronoSeq microfluidic device 100 can sequentially coinject time-tagged beads and cells into the microfluidic chip 110 to generate a droplet at controlled, regular time intervals. Notably, the beads are stored in separate reservoirs (e.g., four examplePCT Patent Application Docket No. 009062.8580.WD00reservoirs Tl , T2, T3, and T4 of the bead reservoirs 130 that correspond to four time points where they are injected with the cells, corresponding to FIG. 2A, 2B, 2C, and 2D, respectively), with each reservoir containing beads with a unique time-tag. For example, for each timepoint shown in FIGS. 2A-2D, the cell sample valve 150 remains open while the corresponding timepoint valve 140 (e.g., orange for Tl, yellow for T2, pink for T3, and green for T4) is opened. This allows for the co-injection of time-specific beads with cells, resulting in libraries barcoded with distinct timetags. This process enables precise temporal labeling of cells across different timepoints. This example embodiment of the ChronoSeq microfluidic device 100 is capable of supporting twelve unique time-tags with 7-minute intervals and 1-minute sampling duration.

[0044] FIG. 2A shows a first timepoint injection. During the first timepoint injection, for example, the cell sample valve 150 is in an open position, allowing cells from the cell reservoir 120 to flow into the microfluidic chip 110 through the cell inlet 111. Simultaneously, a first timepoint valve (TPV 1) is in an open position while the second, third, and fourth timepoint valves (TPV 2, TPV 3, and TPV 4, respectively) are in a closed position. This configuration allows Tl time-tagged beads from a first bead reservoir of the bead reservoirs 130 to be injected into the microfluidic chip 110 through the bead inlet 113, thereby co-encapsulating cells with beads having a first unique time-tag.

[0045] FIG. 2B shows a second timepoint injection. During the second timepoint injection, for example, the cell sample valve 150 remains in an open position, allowing cells from the cell reservoir 120 to continue flowing into the microfluidic chip 110. The first timepoint valve (TPV 1) is now in a closed position, and the second timepoint valve (TPV 2) is in an open position while the third and fourth timepoint valves (TPV 3 and TPV 4) remain in a closed position. This configuration allows T2 time-tagged beads from a second bead reservoir of the bead reservoirs 130 to be injected into the microfluidic chip 110 through the bead inlet 113, thereby coencapsulating cells with beads having a second unique time-tag.

[0046] FIG. 2C shows a third timepoint injection. During the third timepoint injection, for example, the cell sample valve 150 remains in an open position. The first and second timepoint valves (TPV 1 and TPV 2) are in a closed position, and the third timepoint valve (TPV 3) is in an open position while the fourth timepoint valve (TPV 4) remains in a closed position. This configuration allows T3 time- tagged beads from a third bead reservoir of the bead reservoirs 130 to be injected into the microfluidic chip 110 through the bead inlet 113, thereby co-encapsulatingPCT Patent Application Docket No. 009062.8580.WQ00cells with beads having a third unique time-tag.

[0047] FIG. 2D shows a fourth timepoint injection. During the fourth timepoint injection, for example, the cell sample valve 150 remains in an open position. The first, second, and third timepoint valves (TPV 1, TPV 2, TPV 3) are in a closed position, and the fourth timepoint valve (TPV 4) is in an open position. This configuration allows T4 time-tagged beads from a fourth bead reservoir of the bead reservoirs 130 to be injected into the micro fluidic chip 110 through the bead inlet 113, thereby co-encapsulating cells with beads having a fourth unique time-tag.

[0048] The process provides co-injection of time-specific beads with cells, thereby resulting in an output of barcoded cells with distinct time-tags. The process enables precise temporal labeling of cells across different timepoints. .Although FIGS. 2A-2D illustrate four timepoint injections, it should be understood that additional timepoint injections may be performed using additional bead reservoirs containing beads with additional unique time-tags.

[0049] FIG. 3 shows a diagram depicting an example embodiment of a ChronoSeq Library Preparation workflow. The exemplary workflow includes:

[0050] (1) Bead Structure: The beads contain cell barcodes and unique molecular identifiers (UMIs). The exemplary ChronoSeq beads also have an additional Time-Tag common for each Bead Reservoir.

[0051] (2) Droplet generation: Cells and time-tagged beads are co-injected into a microfluidic chip that uses inertial ordering to co-encapsulate one bead with one cell in each droplet, e.g., as discussed in the example shown in FIGS. 2A-2B.

[0052] (3) and (4) Cell Lysis and Molecular Capture: Upon droplet formation, cells are lysed, releasing their mRNA. These are captured by the PolyT region on the beads.

[0053] (5) Sample processing: Droplets are collected and preserved (e.g., kept on ice) until all time points are sampled. Droplets are then broken, and beads are combined, followed by a combined reverse transcription reaction.

[0054] (6) Library preparation: an RNA-Seq library is prepared. This is then followed by Tagmentation and sequencing adapter addition.

[0055] (7) and (8) Sequencing and Data Analysis: After sequencing, a Digital data matrix is created. Time-tags are assigned to each cell barcode, allowing temporal analysis of gene expression.

[0056] The exemplary workflow shown in FIG. 3 includes integrated processes for capturingPCT Patent Application Docket No. 009062.8580.WQ00and analyzing gene expression dynamics across multiple time points. In process (1), time-tagged beads are provided or produced. In some embodiments, for example, the beads are configured to contain cell barcodes and unique molecular identifiers (UMIs). The beads can also be configured to include an additional time-tag that is common for each bead reservoir of the bead reservoirs 130. In some embodiments, for example, each bead reservoir contains beads with a unique timetag, such that beads from different reservoirs can be distinguished based on their respective timetags.

[0057] In process (2), droplet generation is performed. Cells and time-tagged beads are coinjected into the microfluidic chip 110 of an example embodiment of the ChronoSeq microfluidic device 100, e.g., which can be in accordance with the process shown in FIGS. 2A-2D. In some implementations, the microfluidic chip 110 may use inertial ordering to co-encapsulate one bead with one cell in each droplet. This co-encapsulation process results in droplets containing a single cell paired with a single time-tagged bead.

[0058] In process (3) and process (4), cell lysis and molecular capture are performed. The diagram illustrating process (3) shows a top view of the flow-focusing junction depicting the intermixing of cells and beads controllably gated into the junction, which subsequently mixes with injected oil at a downflow region of the flow-focusing junction. The diagram illustrating process (4) shows an enlarged illustration of cells in the oil-fluid medium being lysed such that the released RNA (e.g., mRNA) becomes attached to the time-tagged beads. For example, upon droplet formation, cells within the droplets are lysed, releasing their mRNA, and the released mRNA is then captured by a PolyT region on the time-tagged beads. This capture process enables the association of cellular mRNA with the cell barcode, UMI, and time-tag present on the bead. In some implementations of the process (3), for example, the cell lysis can be implemented in the flow-focusing junction prior to collection, where the lysis of the cells occurs within the droplets after co-encapsulation with the time-tagged beads to release nucleic acid material (e.g., mRNA) from the cells (and capturing the released mRNA on the time-tagged beads through molecular binding with a polynucleotide region on the time-tagged beads). Yet, in some implementations, the cell lysis process can be initiated and / or occur inside the cell reservoir 120, e.g., depending on a type or concentration of detergent used, which can thereby inject lysed or partially lysed cell matter into the flow-focusing junction to co-encapsulate with the time-tagged beads. Moreover, in some implementations of the process (3), the droplets can be heated to assist in inducing lysis.PCT Patent Application Docket No. 009062.8580.WD00

[0059] In process (5), sample processing is performed. Droplets are collected and preserved, for example, by keeping the droplets on ice, until all time points have been sampled. Once all time points are sampled, the droplets are broken (e.g., separation of substances of the emulsion droplets), and beads from the various time points are combined. For example, the demulsification of the droplets enables the beads with the captured genomic material to be processed together as if it were a bulk assay, e.g., even though the individual beads have mRNA from single-cells. A combined reverse transcription reaction is then performed on the combined beads to generate complementary DNA (cDNA) from the captured mRNA. For example, the reverse transcription reaction is performed on these pooled beads, albeit not necessary to be implemented in this order. In some embodiments of the process (5), for example, the reverse transcription can be done before breaking of the droplets when the enzymes and reagents are provided in the cell or bead buffers. The diagram illustrating process (5) shows an example reverse transcription process where the bound mRNA is used the template to synthesize to synthesize complementary DNA (cDNA), e.g., thereby creating stable cDNA from unstable mRNA, which can allow users to study gene expression, clone genes, and / or detect RNA viruses, among other applications.

[0060] In process (6), library preparation is performed. For example, an RNA-Seq library is prepared from the cDNA. This process may include tagmentation and sequencing adapter addition to prepare the library for sequencing.

[0061] In process (7) and process (8), sequencing and data analysis are performed. After sequencing the prepared library, a digital data matrix (e.g., Digital Gene Expression (DGE) matrix) is created. Time-tags are assigned to each cell barcode, allowing temporal analysis of gene expression. This enables the identification of gene expression changes across the multiple time points sampled during the experiment.

[0062] The exemplary workflow shown in FIG. 3 enables the capture of gene expression dynamics across multiple time points in a single experiment, providing temporal resolution for single-cell RNA sequencing analysis.

[0063] Example Equipment Design

[0064] FIG. 4A shows a schematic depicting an example embodiment of the ChronoSeq microfluidic device 100 integrated in an example embodiment of a ChronoSeq system 400, in accordance with the present technology.

[0065] The example ChronoSeq system 400 includes a control device 410, which can bePCT Patent Application Docket No. 009062.8580.WQ00embodied by a computer and / or mobile computing device. The control device 410 is in data communication with various components of the system 400, including the components of the ChronoSeq microfluidic device 100, e.g., to control operation of the respective system component. For example, as shown in FIG. 4A, the control device 410 can be configured to be in communication with exemplary (optional) contingents of the cell reservoir 120, such as an optional magnetic stirrer assembly 423 and / or an optional air / pressure regulator 427. For example, the optional magnetic stirrer assembly 423 can include a magnet positioned in the container that contains the suspended cells that is positioned over a magnetically-controlled stage to promote the rotation of the magnet to stir the cell suspension. The air / pressure regulator 427 can include a pressure controller unit to control the gas and / or the pressure into the cell reservoir 120, bead reservoirs 130, and an oil storage repository. In some embodiments, the ChronoSeq system 400 can include an optional cell media control system that includes a media repository in communication with a cell media pressure controller of the pressure controller unit configured for controlling the flushing of cell media into the one or more containers of the cell reservoir 120; and the optional cell media control system includes a vent valve in communication with one or both of the media repository and / or one or more containers of the cell reservoir 120 to control the venting of air in and / or out of the respective repository and / or container(s). The optional vent valve can be in communication with the control device 410 for the control device 410 to control the state of the vent valve, i.e., in the OPEN state or CLOSED state.

[0066] The control device 410 can be configured to be in communication with exemplary (optional) contingents of the bead reservoirs 130, such as an (optional) automated vortex mixer 433, which can keep the beads evenly suspended in their respective containers of the bead reservoirs 130 before their injection into the ChronoSeq microfluidic device 100. For example, in various implementations, the (optional) automated vortex mixer may provide advantages over magnetic stirrers by reducing dead volumes and simplifying automation for multiple time-tags. In some implementations of the system 400, loading and unloading the beads in their respective containers of the bead reservoirs 130 can be simplified based on the use of tube containers to contain the respective beads (e.g., 50 mL tube), which can be directly attached to the bead reservoir 130. The control device 410 can be configured to be in communication with the cell sample valve 150 and the timepoint valves 140 to control the opening and closing of these respective valves during sequential timepoint injections. In some embodiments of the ChronoSeq system 400, forPCT Patent Application Docket No. 009062.8580.WO00example, the cell sample valve 150 and the timepoint valves 140 can be configured on a valve controller device, e.g., valve controller 405 as illustrated in the diagram of FIG. 4A. The valve controller device can include a plurality of valves and control logic to actuate the opening and closing of the valves, e.g., allowing consolidation of the separately controlled one or more cell sample valves and bead timepoint valves on a single device for enabling miniaturization of the system.

[0067] As further shown in FIG. 4A, in some embodiments, the ChronoSeq microfluidic device 100 can include a second valve controller (valve controller 409). The control device 410 can be configured to be in data communication with a second valve controller 409. The valve controller 409 includes a cell bypass valve 491 and / or a bead bypass valve 493. The cell bypass valve 491 is positioned downstream of the cell sample valve 150 in the fluid path between the cell reservoir 120 and the microfluidic chip 110. The cell bypass valve 491 can be controlled to switch between an ON state or an OFF state, where the OFF state provides the cell sample to the microfluidic chip 110 via the cell inlet 111 for co-encapsulation with time-tagged beads, and the ON state provides the cell sample to a cell waste repository (shown in FIG. 4A as containing bleach to terminate cells in unused cell samples, e.g., during a flushing phase to clear unused cells from the fluid lines without introducing them into the microfluidic chip 110). The bead bypass valve 493 is positioned downstream of the timepoint valves 140 in the fluid path between the bead reservoirs 130 and the microfluidic chip 110. The bead bypass valve 493 can be controlled to switch between an ON state or an OFF state, where the OFF state provides the particular time-tagged beads (selected by a particular timepoint valve of the timepoint valves 140) to the microfluidic chip 110 via the bead inlet 113 for co-encapsulation with cells, and the ON state provides the particular time-tagged beads to a time-tagged beads recovery repository 460 (e.g., for example, to recover unused beads from the fluid lines during a flushing phase, thereby minimizing waste and reducing experimental costs).

[0068] In some embodiments, for example, the ChronoSeq microfluidic device 100 includes a cell channel flow meter 170 positioned in the flow channel between the cell sample valve 150 and the cell bypass valve 491. The cell channel flow meter 175 can be configured to measure the flow rate of cells through the cell channel. The control device 410 can be configured to be in communication with the cell channel flow meter 170, e.g., enabling the computer to monitor and control the flow rate of cells during both flushing and injection phases. In some embodiments, forPCT Patent Application Docket No. 009062.8580.WQ00example, the ChronoSeq microfluidic device 100 includes a bead channel flow meter 175 positioned in the flow channel between the timepoint valves 140 and the bead bypass valve 493. The bead channel flow meter 175 is configured to measure the flow rate of time-tagged beads through the bead channel. The control device 410 can be configured to be in communication with the bead channel flow meter 175, e.g., enabling the computer to monitor and control the flow rate of beads during both flushing and injection phases. The cell channel flow meter 170 and the bead channel flow meter 175 can provide the ChronoSeq microfluidic device 100 and the ChronoSeq system 400 with precise control needed for high-quality droplet formation during low flow rate operation, while also enabling the higher flow rates used during flushing phases.

[0069] As further shown in FIG. 4A, the ChronoSeq system 400 can (optionally) include one or more multi-axes robots to control precise positioning of the outputs, i.e., output of the microfluidic chip 110 and / or output of the excess (unused) time-tagged beads. For example, the one or more multi-axes robots can include a 2-Axis XY robot and a 3-Axis XYZ robot, each in data communication with the control device 410. In some embodiments, the example 2-Axis XYZ robot can include one or more stage(s) operatively coupled to one or more X-axis motor(s) and one or more Y-axis motor(s), that operate in conjunction, to provide two-dimensional motion of the one or more stage(s). In some embodiments, the example 3-Axis XYZ robot can include one or more stage(s) operatively coupled to one or more XY-axes motor(s) and one or more Z-axis motor(s), that operate in conjunction, to provide multiple dimensional motion of the one or more stage(s).

[0070] As illustrated in the diagram of FIG. 4A, an exemplary 2-Axis XY robot is configured to control the positioning of the droplets output from the outlet 119 of the microfluidic chip 110 (e.g., via outlet tubing from the microfluidic chip 110). The 2-Axis XY robot can automatically move the outlet tubing to a correct collection tube, e.g., preventing the mixing of droplets from different timepoints with washing fluids. Example collection tubes can include a waste tube (e.g., to collect waste fluid routed via a bead bypass valve associated with the example 3-Axis XYZ robot), a good collection tube (e.g., to collect the outputted droplets), and a bad collection tube (e.g., to collect waste fluid flow from the microfluidic chip 110, e.g., during the washing / flushing phase), which the control device 410 can determine the proper output dispensing based on diagnostics assessments of the system 400 in its implementations. This robotic system facilitates droplet collection by ensuring that droplets containing co-encapsulated cells and time-tagged beadsPCT Patent Application Docket No. 009062.8580.WQ00from each timepoint are directed to the appropriate collection vessel. In some embodiments, the droplets are collected in tubes kept on ice to preserve sample integrity until all time points are sampled. Also, as illustrated in the diagram of FIG. 4A, an exemplary 3-Axis XYZ robot is configured to facilitate bead recovery from the fluid lines. For example, when the bead bypass valve 493 is in an ON state during a flushing phase, unused time-tagged beads may be diverted from the fluid path leading to the microfluidic chip 110, and the- 3 Axis XYZ robot is configured to position collection vessels or tubing to recover these unused beads, minimizing waste and reducing experimental costs. The additional axis of movement provided by the 3-Axis XYZ robot may enable more precise positioning for bead recovery operations, e.g., as compared to the 2-Axis XY robot used for droplet collection. Notably, a 3-Axis XYZ robot may be used (instead of the 2-Axis XY robot) to controllably manage output droplet collection from the microfluidic chip 110 in some embodiments. The control device 410 coordinates the operation of the exemplary 2-Axis XY robot and / or 3-Axis XYZ robot in conjunction with the valve controllers, flow meters, and other components of the ChronoSeq microfluidic device 100 to enable automated, controlled, sequential introduction of cells and time-tagged beads while maintaining sample isolation and preventing cross-contamination between timepoints.

[0071] As further shown in FIG. 4A, the ChronoSeq microfluidic device 100 includes an oil channel flow meter 180 positioned in an oil flow channel between an oil pressure controller (of the optional air / pressure regulator 427) and an oil bypass valve (e.g., configured on the valve controller 405). The oil bypass valve can be controlled by the control device 410 via the valve controller 405 and is configurable between an ON state and an OFF state. For example, when the oil bypass valve is in an OFF state, oil from an oil storage repository is caused to flow. e.g. via the oil pressure controller, through the oil channel to the microfluidic chip 110 via an oil inlet 115. The oil is used to intermix with the cell / bead fluid medium in the flow-focusing junction to generate droplets, where cells and time-tagged beads are co-encapsulated in oil droplets. When the oil bypass valve is in an ON state, oil is diverted away from the microfluidic chip 110 and directed to an oil waste repository configured to collect the unused oil for reuse, thereby reducing waste and operational costs. The oil channel flow meter 180 is configured to measure the flow rate of oil through the oil channel and is in data communication with the control device 410. The control device 410 can control coordination of the operations of the oil pressure controller, the oil channel flow meter 180, and the oil bypass valve to enable precise control of oil flow during bothPCT Patent Application Docket No. 009062.8580.WQ00flushing and injection phases, for precise droplet formation. The oil pressure controller enables rapid changes in flow rate, while the oil channel flow meter provides the precise control needed for high-quality droplet formation during low flow rate operation. Together with the cell channel flow meter 170 and the bead channel flow meter 175, the oil channel flow meter 180 enables the ChronoSeq microfluidic device 100 to achieve the flow control necessary for reliable droplet generation across multiple timepoint injections.

[0072] The example embodiment of the ChronoSeq microfluidic device 100 and ChronoSeq system 400 addresses several challenges not present in conventional systems, such as a Drop-seq system, which include (1) extended cell viability, (2) cross-contamination prevention, and (3) sample isolation. For example, the disclosed ChronoSeq devices and systems maintain cell suspension viability for experiments lasting two hours or longer, e.g., compared to typical 30-minute Drop-seq runs. For example, the disclosed ChronoSeq devices and systems can provide a washing system to remove unused cells or beads between samples. For example, the disclosed ChronoSeq devices and systems are structurally designed to prevent the mixing wash fluid with droplets.

[0073] The disclosed ChronoSeq devices and systems device can be configured to operate in two main phases during each injection cycle: a flushing phase that clears fluid lines of residual beads or cells from previous injections; and an injection phase that precisely introduces cells and time-tagged beads into the microfluidic chip 110. This integrated system enables controlled, sequential introduction of cells and time-tagged beads, capturing gene expression dynamics across multiple time points in a single experiment. The disclosed design prioritizes cell viability maintenance, precise fluid control, and efficient bead usage, all critical for generating high-quality, time-resolved single-cell sequencing data.

[0074] The ChronoSeq system 400 can be configured to process omics data according to a variety of methods. In some embodiments, a ChronoSeq data processing method includes assignment process involving extraction and assignment of the time-tag to each cell barcode associated with the collected droplet. For example, the process can include searching for the sequence TTTTT<Time-Tag>TTTTT in a first read, e.g., in the region after the UMI on the bead. For each cell barcode, the number of occurrences of each time-tag can be counted (e.g., twelve unique time-tags in total for some embodiments). The time-tag with more than 70% of the counts can be assigned to that cell barcode.PCT Patent Application Docket No. 009062.8580.WO00

[0075] In some embodiments, the ChronoSeq data processing method can include, prior to the assignment process, a data pre-processing technique to perform quality control on the raw data. In some embodiments, the ChronoSeq data processing method can include, after the assignment process, a conversion process to convert the assignment-processed data from single-cell droplets into a digital gene expression (DGE) matrix. In some embodiments, a ChronoSeq data processing method can include a data alignment process can include aligning the reads to a reference genome, e.g., such as using the STAR aligner.

[0076] FIG. 4B shows a diagram of an example embodiment of the control device 410 implemented in various example embodiments of the ChronoSeq system 400. The control device 410 may be embodied by a computer or computing device, e.g., in a standalone configuration or in a network of computers in a data processing system configuration. The control device 410 may be embodied by a mobile communication device (e.g., smartphone, tablet, smartwearable, etc.).

[0077] In various embodiments, the control device 410 can include a data processing unit, which comprises one or more processor(s) to process data, one or more memory unit(s) in communication with the processor(s) to store data, and an input / output unit (RO) to interface the processor(s) and / or memory unit(s) to other modules, units or devices of the control device 410 or external devices. For example, the processor(s) can include a central processing unit (CPU), a microcontroller unit (MCU), a graphics processing unit (GPU), or other processing unit. For example, the memory unit(s) can include and store processor-executable code, which when executed by the processor(s), configures the data processing unit to perform various operations, e.g., such as receiving information, commands, and / or data, processing information and data, and transmitting or providing information / data to another device. In some implementations, the data processing unit can transmit raw or processed data to a computer system or communication network accessible via the Internet (‘the cloud’) that includes one or more remote computational processing devices (e.g., servers in the cloud). To support various functions of the data processing unit, the memory unit(s) can store information and data, such as instructions, software, values, images, and other data processed or referenced by the processor(s). For example, various types of Random Access Memory (RAM) devices, Read Only Memory (ROM) devices, Flash Memory devices, and other suitable storage media can be used to implement storage functions of the memory unit(s).

[0078] In some embodiments, the control device 410 can include a wireless communicationPCT Patent Application Docket No. 009062.8580.WD00unit. For example, in some implementations, the I / O of the data processing unit can interface the data processing unit with the wireless communications unit to utilize various types of wired or wireless interfaces compatible with typical data communication standards, for example, which can be used in communications of the data processing unit with other devices, via a wireless transmitter / receiver (Tx / Rx) unit, e.g., including, but not limited to, Bluetooth, Bluetooth low energy (BLE), Zigbee, IEEE 802.11, Wireless Local Area Network (WLAN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access (WiMAX)), 3G / 4G / LTE / 5G / 6G cellular communication methods, NFC (Near Field Communication), and parallel interfaces.

[0079] The I / O of the data processing unit can also interface with other external interfaces, sources of data storage, and / or visual or audio display devices, etc. to retrieve and transfer data and information that can be processed by the processor(s), stored in the memory unit(s), or exhibited on an output unit of the control device 410 or an external device. For example, in some embodiments, the control device 410 can optionally include a display unit configured to be in data communication with the data processing unit, e.g., via the TO, to provide a visual display, an audio display, and / or other sensory display that produces a user interface of a software application in accordance with the mobile manufacturing system. In some examples, the display unit can include various types of screen displays, speakers, or printing interfaces, e.g., including but not limited to, light emitting diode (LED), or liquid crystal display (LCD) monitor or screen, cathode ray tube (CRT) as a visual display; audio signal transducer apparatuses as an audio display; and / or toner, liquid inkjet, solid ink, dye sublimation, inkless (e.g., such as thermal or UV) printing apparatuses, etc.

[0080] FIG. 5 shows a diagram depicting an example implementation comparing an example embodiment of the ChronoSeq microfluidic device 100 and ChronoSeq system 400 (shown in upper panel (A)) and a conventional Drop-seq device (shown in lower panel (B)).

[0081] The exemplary ChronoSeq microfluidic device 100 is controlled, for example, via serial connections to the control device 410 (e.g., a computer in some embodiments), which coordinates the actions of its various components. In contrast, as illustrated in FIG. 5, the conventional Drop-seq device works by setting flow rates manually on syringe pumps, e.g., cell syringe pump 510. bead syringe pump 520, and oil syringe pump 530.

[0082] Some exemplary distinguishing features of the exemplary ChronoSeq microfluidicPCT Patent Application Docket No. 009062.8580.WQ00device 100 include the following.

[0083] 1. Bead handling: The exemplary ChronoSeq microfluidic device 100 allows for the easy addition of more time-tags as necessary or desired. Additionally, loading and unloading the beads is simplified, e.g., as a new container tube (e.g., 50 mLtube) containing beads can be directly configured into the reservoir. Moreover, the time-tagged bead reservoirs are connected to timepoint valves, which facilitate the selection of the appropriate time — tagged beads for injection into the microfluidic chip. Drop-seq lacks each of these capabilities. Also, for some embodiments, the exemplary ChronoSeq microfluidic device 100 can incorporate an automated vortex mixer to keep the beads evenly suspended before injection. In contrast, Drop-seq uses a magnetic stirrer for bead suspension, which leads to high dead volumes and complicates automation for multiple time-tags.

[0084] 2. Cell culture system: In conventional Drop-seq devices, cells are directly loaded into a syringe for injection, e.g., typically running for 15 to 30 minutes. However, a key goal of the disclosed technology is to maintain cell viability for at least 2 hours. Leaving cells at room temperature without growth media and in the absence of 5% CO2 can lead to excessive cell death and unwanted perturbations. Thus, the exemplary ChronoSeq microfluidic device 100 includes an integrated a cell culture system directly into the ChronoSeq device, e.g., featuring a water bath to maintain cells at 37°C, along with a 5% CO2 gas cylinder that serves as a pressure source and aids in cell culture. Furthermore, additional cell containers (e.g., tubes) can be added to the cell reservoir 120 to culture multiple cell suspensions separately and use separate corresponding valves (in the cell sample valve 150 contingent) to select which cell suspension is injected into the microfluidic chip 110.

[0085] 3. Droplet collection: In conventional Drop-seq devices, droplets are collected directly in an outflow container 550, e.g., such as a 50 mL tube, kept on ice. In contrast, in the exemplary ChronoSeq microfluidic device 100. beads and cells from previous injections are crucially prevented from mixing with those from new injections. In some embodiments of the ChronoSeq microfluidic device 100, for example, the ChronoSeq microfluidic device 100 includes a washing system to clear the fluid lines of residual beads and cells. Moreover, the exemplary ChronoSeq microfluidic device 100 ensures that washing liquids and unused cell suspensions do not mix with the collected droplets. In some embodiments of the ChronoSeq microfluidic device 100, for example, the ChronoSeq microfluidic device 100 includes a robotic system that facilitates dropletPCT Patent Application Docket No. 009062.8580.WD00collection and bead recovery. This exemplary robotic system automatically moves the outlet tubing to the correct tube, preventing the mixing of droplets with washing fluids. Additionally, it can recover unused beads from the fluid lines, minimizing waste and reducing experimental costs.

[0086] 4. Flow control: In conventional Drop-seq devices, precise speed control is essential for droplet formation. Speeds that are too high can inhibit droplet formation, while low speeds can negatively impact throughput. For instance, like Drop-seq. exemplary ChronoSeq devices can provide cell and bead flow rates of 10 pL / min to 70 pL / min. Yet, to achieve precise control, Drop-seq employs syringe pumps, which allow direct manipulation of the syringe’s squeezing rate. However, in contrast, the ChronoSeq microfluidic device 100 can switch between the lower flow rates for cell / bead flow to high flow rates for flushing, e.g., of 1,000 to 3,000 pL / min. Low flow rates, like Drop-seq flow rates for washing, are insufficient for quickly washing unused beads and cells from its device. Notably, for example, some embodiments of the ChronoSeq microfluidic device 100 are configured with wide channels of the flow resistors that prevent blockage, e.g., in contrast with the narrow channels (e.g., 60 pm width or less) employed by the conventional Drop-seq devices. Moreover, in some embodiments, the ChronoSeq microfluidic device 100 can include a compressed air pressure controller and flow sensors for each of the bead, cell, and dispersion fluid (e.g., oil and / or air flow) channels. The compressed air controller enables rapid changes in flow rate, while the flow sensors provide the precise control needed for high-quality droplet formation during low flow rate operation. For example, the compressed air controller can be combined with a PID (proportional-integral derivative) control. Notably, pressure-driven flow control is superior to syringe pumps for rapid changes in flow rate, e.g., as pressure systems can adjust flow in milliseconds; whereas syringe pumps often take minutes due to mechanical inertia and system compliance.

[0087] Example Microfluidic Chip Design

[0088] A primary function of the microfluidic chip 110 of the ChronoSeq microfluidic device 100 is to co-encapsulate cells with time-tagged beads using a flow-focusing junction at the center of the chip. Like the overall system design of conventional systems, such as Drop-seq, current omics systems are ill equipped facilitate co-encapsulation with precision to ensure accuracy and efficiency of the cell-tagged samples. These limitations are addressed and solved by the disclosed ChronoSeq technology.

[0089] Some example distinguishing features of the exemplary microfluidic chip 110 of thePCT Patent Application Docket No. 009062.8580.WQ00ChronoSeq microfluidic device 100 include the following.

[0090] 1. Ordering mechanism. Conventional Drop-seq devices rely on random (e.g., Poisson) ordering for co-encapsulation of cells and beads. This random ordering makes it difficult to increase the number of cells collected per timepoint without also increasing droplets containing multiple cells or beads. In contrast, the microfluidic chip 110 of the exemplary ChronoSeq microfluidic device 100 includes a deterministic ordering mechanism. Particularly, the microfluidic chip 110 has an optimized inertial ordering design engineered to increase cell capture through deterministic ordering of beads into droplets. This deterministic ordering allows for capturing more cells by increasing the number of droplets containing exactly one bead and one cell.

[0091] 2. Channel design. Both the Drop-seq chip and existing inertial ordering designs use narrow flow resistance channels, which are prone to blockage during the flushing phase. In contrast, the microfluidic chip 110 includes channels configured to reduce blockage risk, e.g., including widened channels in the chip. In some embodiments, for example, the channels of the microfluidic chip 110 can be configured to be 120 pm wide and 100 pm high; and in some embodiments the channels can be configured in a range of 80 pm to 160 pm in width and 80 pm to 120 pm in height. In some embodiments, for example, the channels of the microfluidic chip 110 can be configured to have a length in a range of 50 mm to 200 mm; and in some embodiments the length of the channels can be around 125 mm. Such configurations are far capable of providing a microparticle (e.g., bead) flow rate greater than an estimated minimum bead flow rate of ~55 pL / min to maintain the deterministic ordering based on the exemplary inertial ordering design of the microfluidic chip 110. Moreover, the channel design of the microfluidic chip 110 is configured to maintain the same flow resistance but with wider channels, e.g., via an increased the length of the channels to compensate for the flow resistance, e.g., with width / height to length aspect ratios of around 100:1 to about 120:1.

[0092] For manufacturing, standard photolithography and PDMS molding techniques can be used for chip fabrication of the microfluidic chip 110. For example, in some embodiments, batches of the microfluidic chip 110 can be produced by plasma bonding to glass slides and chemically treating to create hydrophobic channels. The fabrication techniques allow for rigorous quality control protocols, e.g., including compressed air testing to check for delamination and eliminate leaking chips, simulating the high pressures used during the flushing phase; visual inspection toPCT Patent Application Docket No. 009062.8580.WQ00ensure the absence of particles before connecting to the device. These design and quality control measures enable the chips to withstand higher pressures and cyclical pressurization during the flushing phase. The optimized chip design is crucial for reliable, high-throughput, time-resolved single-cell sequencing. The improvements in channel geometry and manufacturing process contribute to reduced clogging, increased cell capture efficiency, overall system robustness. These features are essential for maintaining consistent performance across multiple time points and ensuring high-quality data collection throughout extended experimental runs.

[0093] FIGS. 6 A and 6B show diagrams depicting an example embodiment and implementation of a ChronoSeq microfluidic chip, in accordance with the present technology. FIG. 6 A shows an example embodiment of the microfluidic chip 110 design. FIG. 6B shows an example side-by-side comparison of the chip designs of the example embodiment of the microfluidic chip 110 of FIG. 6A next to a chip design of a conventional Drop-seq chip.

[0094] As shown in FIG. 6A, the exemplary microfluidic chip 110 includes a substrate 101, on or in which are flow channel systems. In some embodiments, the flow channel systems are configured as channel indentations etched into the substrate 101 and covered via a cover on top of the substrate 101; whereas in some embodiments, the flow channel systems are configured as tubes that are fabricated on top of the substrate 101. In various embodiments, the channel systems can be configured as cylindrical or rectangular channels. In some implementations, for example, the microfluidic chip 110 can fabricated by CNC machining, injection molding, or other microfabrication techniques.

[0095] The exemplary micro fluidic chip 110 includes a cell flow channel system 102 that is coupled to the cell inlet 111; a bead flow channel system 104 that is coupled to the bead inlet 113, and an oil flow channel system 106 that is coupled to the oil inlet 115. Each of the cell flow channel system 102, the bead flow channel system 104, and the oil flow channel system 106 flow their respective substances from the inlets to the flow-focusing junction region where the intermixing occurs, e.g., previously described above. Notably, in some embodiments of the microfluidic chip 110. the flow-focusing junction region includes to two (or more) separate cell flow channels of the cell flow channel system 102 that converge to meet one (or more) bead flow channel of the bead flow channel system 104, which together form a single channel to which two (or more) separate oil flow channels of the oil flow channel system 106 converges, which then leads to outlet 119. An example of the flow-focusing junction structure is illustrated in FIG. 3 inPCT Patent Application Docket No. 009062.8580.WO00connection with the process (3).

[0096] The exemplary design of the microfluidic chip 110 has several advantages over conventional microfluidic-based omics devices and systems. FIG. 6B juxtaposes an example embodiment of the microfluidic chip 110 of the ChronoSeq microfluidic device 100 next to a conventional Drop-seq chip. As one example advantage, with respect to the Drop-seq chip, the microfluidic chip 110 of the exemplary ChronoSeq microfluidic device 100 does not require a mixer contingent (such as the droplet mixer of a Drop-seq chip, which is near the Drop-seq outlet hole), which substantially reduces any likelihood of blockage — a common problem for Drop-seq devices. Also, as another example advantage, the micro fluidic chip 110 of the exemplary ChronoSeq microfluidic device 100 includes flow channels engineered to optimal flow resistance, e.g.. modifying width and length configurations of the flow channels. Yet even further, as another example advantage, the microfluidic chip 110 of the exemplary ChronoSeq microfluidic device 100 includes an inertial (deterministic) ordering spiral design, which is in contrast with the straight (Poisson) ordering inlet of a Drop-seq chip.

[0097] These exemplary advantageous features of the disclosed ChronoSeq devices and systems substantially improve the performance of the microfluidic chip 110 in terms of reduced clogging, maintained flow dynamics, and enhanced cell / bead ordering for more efficient singlecell encapsulation, for example. The inertial ordering design, in particular, allows for a more deterministic approach to cell and bead pairing, potentially increasing the capture efficiency of single cells with single beads.

[0098] Example Time-Tagged Beads Design and Validation Implementations

[0099] Example implementations of the disclosed ChronoSeq devices and systems were performed to validate the accuracy, robustness, effectiveness, and efficiency of the present technology.

[0100] FIG. 7 shows a diagram depicting an example bead modification workflow for creating ChronoSeq Time-Tagged beads. The workflow can begin with a bead having an attached oligonucleotide (oligo), sometimes referred to as a “sticker” oligo. The initial oligo attached to the bead includes a PCR handle, a cell barcode, a unique molecular identifier (UMI), and a PolyT region (a short, synthetic sequence of consecutive thymine (T) nucleotides). For example, the PCR handle enables amplification of captured sequences during library preparation; the cell barcode provides a unique identifier for each bead, e.g., enabling the association of captured mRNA withPCT Patent Application Docket No. 009062.8580.WQ00a specific cell; the UMI provides a unique identifier for each captured mRNA molecule, e.g., enabling the identification and removal of PCR duplicates during data analysis; and the PolyT region is configured to capture mRNA released from lysed cells by hybridizing to the polyadenylated (PolyA) tail of the mRNA.

[0101] The workflow can continue with the bead undergoing one or more modification steps to add a time-tag sequence. For example, the modification process involves using another oligo having a PolyA region (a short, synthetic sequence of consecutive adenine (A) nucleotides) that binds to the PolyT region on the original oligo of the bead. Notably, the other oligo includes a Time Tag sequence sandwiched between two PolyA regions (e.g., a PolyA +A10 and a PolyA +A20). The 3' end of the bead-attached oligo is then extended to the reverse complement of the bound oligo using a DNA polymerase, such as E. coli DNA Polymerase I. Following extension, alkaline denaturation can be used to make the double-stranded DNA single- stranded, thereby exposing the newly added sequences. The resulting modified bead, as shown in FIG. 7, thus includes the original PCR handle, cell barcode, and UMI, along with two flanking sequences with a time-tag positioned between them. The time-tag is a unique sequence that is common for all beads within a given bead reservoir, but configured to differ with other time-tags on beads to be contained in other bead reservoirs of the bead reservoirs 130. In some embodiments of the timetag beads, the Time-Tag can be directly synthesized onto the microparticle (e.g., bead), which can produce the time-tag beads without use of the sticker oligo. In this manner, instead of using the PolyT region, the Time-Tag is configured before the polyT region of the beads after the UMI (not shown in FIG. 7). Other configurations of the Time-Tag are possible, e.g., the position of the time barcode on the microparticle is not critical as long as the Time-Tag includes a unique value (e.g., UMI).

[0102] Example time-tagged RNA-seq beads for use in example implementations of the disclosed ChronoSeq devices and systems were produced for validation experiments. For example, these beads were used in bulk RNA-Seq experiments (FIG. 8) and single cell RNA-Seq experiments, e.g., using an example embodiment of the ChronoSeq microfluidic device 100 for the single cell RNA-Seq experimental implementations.

[0103] The Bulk RNA-Seq validation experiments were performed by directly mixing human cells (K562) with odd numbered time-tags and mouse cells (EL4) with even numbered time-tags. The example data, shown in FIG. 8, demonstrated clear separation of time-tags with very littlePCT Patent Application Docket No. 009062.8580.WQ00cross-contamination.

[0104] The Single Cell RNA-Seq validation experiments were performed by co-injecting 50:50 human-mouse mixed, human only and mouse only suspensions with the first, second, and third time-tags, respectively. The time-tags can separate each injection uniquely, meaning there are almost no time-tags from the previous injection and no cross-contamination. Moreover, the time-tags capture only the cells co-injected with them, showing effective temporal barcoding of cells. Next, the data generated in the first time-tag has very few mixed cell barcodes from humans and mice, e.g., meaning human and mouse cells are uniquely captured with low noise, comparable to other technologies.

[0105] FIG. 8 shows data plots depicting example results of a bulk RNA-Seq validation implementation of twelve time-tags. For example, 10 pL of Time-Tagged beads at 450 beads / pL suspended in Lysis Buffer were directly mixed with 8 pL of Mouse or Human cells at 600cells / pL. Odd numbered Time- Tags were mixed with Human (K562) cells while even numbered Time-Tags were mixed with Mouse (EL4) cells. Each dot in the respective data plot represents a unique Cell Barcode, and the (X, Y) coordinates represent the number of Human and Mouse transcripts respectively. The Cell Barcodes associated with their respective Time- Tags show clear separation according to the species of the Cells they were mixed with.

[0106] FIG. 9 shows data plots depicting example results of a single-cell experiment to verify quality of data generated using the Single-Cell RNA-Seq version of the disclosed ChronoSeq technology. Automated sampling within the exemplary ChronoSeq device showed similarly precise tagging and each cell population was exclusively labeled with its designated barcode. Three separate reservoirs for maintaining cell suspensions were set up inside the example ChronoSeq device. Cells could be sampled from either Reservoir 1, 2 or 3 and co-injected with any of the Time-tagged beads labeled 1-12. A 50:50 mixture of human(K562) and mouse(EL4) cells was added to Reservoir 1, a human only (K562) suspension to Reservoir 2 and a mouse only(EL4) suspension to Reservoir 3. Cells were sampled with a 7-minute sampling interval including a 1-minute sampling duration using the ChronoSeq device. Three samples were taken and barcoded with three separate Time-Tags. The first sample (left plot) was from a 50:50 Human (K562) and Mouse (EL4) Cell Suspension. The second sample (center data plot) was from a Human only cell suspension. While the third sample (right data plot) was from a Mouse only cell suspension. Each dot in the three plots represents a Unique Cell Barcode and the (X, Y)PCT Patent Application Docket No. 009062.8580.WO00coordinates represent the number of Human and Mouse transcripts respectively. Cells were injected at a concentration of 215 cells / pL while the bead concentration was 450 beads / pL. . The first, second and third cell suspensions were sequentially co-injected with Time-Tags 10 through 12. Species-mixing barnyard plots clearly showed Time-Tag 10 capturing both human and mouse cells, Time-Tag 11 capturing human cells and Time-Tag 12 capturing mouse cells.

[0107] Example Validation Time-Series Data by Measuring Inflammatory Response

[0108] For the RNAseq version of the assay, a preliminary bulk time-series was generated using these beads for K562 cells stimulated with TNFa (lOng / ml).

[0109] FIG. 10 shows data plots depicting example results of example valuation implementations for bulk RNA-seq from K562 cells simulated with TNFa at 15 mins (left) and 65 mins (right) show key time-delays and kinetic differences for gene activation.

[0110] X-axis is time in minutes, and Y-axis is the log normalized fold change with respect to an unperturbed control for several representative genes. Because of the delayed addition of TNFa to the experiment on the right, the activation of these representative genes is delayed compared to the experiment on the left with a shifting pattern. However, both experiments exhibit diverse delay and rate of gene activation. For example, NFKBIA gets activated fastest, ICAM1 next and TNFRSF9 last. This delayed activation of TNFRSF9 indicates that it is not the initiator of the expression change cascade, providing valuable causal insights. Additionally, even genes activated simultaneously can display different activation rates; for example, NFKBIA shows a rapid, steep increase, whereas IL8 demonstrates a more gradual rise. These fine-grained temporal dynamics would be undetectable using conventional technologies, which typically have lower temporal resolution.Example

[0111] In some embodiments in accordance with the present technology (example Al), a method for fast temporal single cell measurements includes: injecting a first sample of cells from a repository into a cell inlet of a microfluidic chip; injecting a first set of time-tagged beads having a different time barcode than other sets of time-tagged beads into a bead inlet of the microfluidic chip; creating a first time-tagged single cell RNA seq library based on producing a first time-tagged sample of cells from the first sample of cells being time-tagged with the first set of time-tagged beads having a corresponding time point: and collecting the first time-tagged sample of cells from an outlet of the microfluidic chip.PCT Patent Application Docket No. 009062.8580.WO00

[0112] Example A2 includes the method of example Al or any of examples A1-A6, further comprising: injecting a second sample of cells into the cell inlet of the microfluidic chip; injecting a second set of time-tagged beads having a different time barcode than the first set of time-tagged beads and other sets of time-tagged beads into the bead inlet of the microfluidic chip; creating a second time-tagged single cell RNA seq library based on producing a second time-tagged sample of cells from the second sample of cells being time-tagged with the second set of time-tagged beads having a corresponding time point; and collecting the second time-tagged sample of cells from the outlet of the microfluidic chip.

[0113] Example A3 includes the method of example A2 or any of examples A1-A6, wherein different sets of time-tagged beads are injected into the microfluidic chip at seven minute or less intervals.

[0114] Example A4 includes the method of example Al or any of examples A1-A6, wherein the collecting takes one minute or less.

[0115] Example A5 includes the method of example Al or any of examples A1-A6, wherein the injecting the first sample of cells includes controlling a valve of a first fluid channel in fluid connection with the microfluidic chip and with the repository containing a plurality of cells.

[0116] Example A6 includes the method of example Al or any of examples A1-A5, wherein the injecting the first set of time-tagged beads includes controlling a first timepoint valve of a plurality of timepoint valves of a second set of fluid channels in fluid communication with the microfluidic chip.

[0117] In some embodiments in accordance with the present technology (example Bl), a method for temporal single-cell genomic sequencing includes providing a microfluidic chip having a cell inlet, a bead inlet, and an outlet; injecting a first sample of cells from a cell reservoir into the cell inlet of the microfluidic chip; injecting a first set of time-tagged beads from a first bead reservoir of a plurality of bead reservoirs into the bead inlet of the microfluidic chip, wherein the first set of time-tagged beads comprises a first time barcode; co-encapsulating cells from the first sample of cells with time-tagged beads from the first set of time-tagged beads in droplets within the microfluidic chip to produce a first time-tagged sample; collecting the first time-tagged sample from the outlet of the microfluidic chip; injecting a second sample of cells from the cell reservoir into the cell inlet of the microfluidic chip; injecting a second set of time-tagged beads from a second bead reservoir of the plurality of bead reservoirs into the bead inlet of the microfluidic chip,PCT Patent Application Docket No. 009062.8580.WO00wherein the second set of time-tagged beads comprises a second time barcode different from the first time barcode; co-encapsulating cells from the second sample of cells with time-tagged beads from the second set of time-tagged beads in droplets within the microfluidic chip to produce a second time-tagged sample; and collecting the second time-tagged sample from the outlet of the microfluidic chip.

[0118] Example B2 includes the method of example Bl or any of examples B1-B13, wherein the injecting the first set of time-tagged beads comprises opening a first timepoint valve associated with the first bead reservoir while maintaining a cell sample valve in an open position and maintaining a second timepoint valve associated with the second bead reservoir in a closed position.

[0119] Example B3 includes the method of example Bl or any of examples B1-B13, further comprising: performing a flushing phase between collecting the first time-tagged sample and injecting the second sample of cells, wherein the flushing phase clears fluid lines of residual beads or cells from the first time-tagged sample.

[0120] Example B4 includes the method of example Bl or any of examples B1-B13, wherein the injecting the second set of time-tagged beads occurs at an interval of seven minutes or less after the injecting the first set of time-tagged beads.

[0121] Example B5 includes the method of example Bl or any of examples B1-B13, wherein each of the collecting the first time-tagged sample and the collecting the second time-tagged sample has a sampling duration of one minute or less.

[0122] Example B6 includes the method of example Bl or any of examples B1-B13, wherein the co-encapsulating comprises using inertial ordering to co-encapsulate one bead with one cell in each droplet.

[0123] Example B7 includes the method of example Bl or any of examples B1-B13. further comprising: injecting one or more additional sets of time-tagged beads from a corresponding one or more additional bead reservoirs of the plurality bead reservoirs into the bead inlet of the microfluidic chip at successive time intervals, wherein each additional set of time-tagged beads comprises a unique time barcode different from all other time barcodes.

[0124] Example B8 includes the method of example B7 or any of examples B1-B13, wherein the method supports at least twelve unique time barcodes.

[0125] Example B9 includes the method of example Bl or any of examples B1-B13, whereinPCT Patent Application Docket No. 009062.8580.WQ00the time-tagged beads comprise a cell barcode, a unique molecular identifier (UMI), a time-tag, and a polynucleotide region configured to capture mRNA released from lysed cells within the droplets.

[0126] Example BIO includes the method of example Bl or any of examples B1-B13, further comprising: prior to the collecting, lysing the cells within the droplets to release mRNA from the cells; and capturing the released mRNA on the time-tagged beads through molecular binding with a polynucleotide region on the time-tagged beads.

[0127] Example B 11 includes the method of example B 10 or any of examples B 1-B 13, further comprising: breaking the droplets after collecting all time-tagged samples; combining beads from the first time-tagged sample and the second time-tagged sample; and performing a reverse transcription reaction on the combined beads.

[0128] Example B12 includes the method of example Bl or any of examples B 1-B 13, further comprising: maintaining the cell reservoir at 37°C and in a presence of 5% CO2 to maintain cell viability throughout a full course of the method.

[0129] Example B13 includes the method of example Bl or any of examples B 1-B 12, further comprising: automatically moving outlet tubing to different collection tubes to prevent mixing of the first time-tagged sample with the second time-tagged sample.

[0130] In some embodiments in accordance with the present technology (example B14), a microfluidic chip for temporal single-cell genomic sequencing includes: a substrate having a plurality of flow channels including a cell flow channel, a microparticle flow channel, and a dispersion fluid flow channel; a cell inlet coupled to the cell flow channel configured to receive cells; a microparticle inlet coupled to the microparticle flow channel configured to receive time-tagged microparticles; a dispersion fluid inlet coupled to the dispersion fluid flow channel configured to receive at least one of oil or air; a flow-focusing channel junction in fluid communication with at least one region of the cell flow channel, the microparticle flow channel, and the dispersion fluid flow channel, wherein the flow-focusing junction is configured to receive a first fluid medium containing the cells, a second fluid medium containing the time-tagged microparticles, and the at least one of oil or air and to co-encapsulate cells with time-tagged microparticles in droplets, wherein the microparticle flow channel is configured as an inertial ordering channel comprising a spiral design to provide deterministic ordering of the time-tagged microparticles into the flow-focusing channel junction; and an outlet configured to output dropletsPCT Patent Application Docket No. 009062.8580.WQ00containing co-encapsulated cells and time-tagged microparticles.

[0131] Example B15 includes the microfluidic chip of example B14 or any of examples B14-B19, wherein the inertial ordering channel is configured to provide deterministic ordering of the microparticles to increase a number of droplets containing one microparticle and one cell.

[0132] Example B 16 includes the microfluidic chip of example B 14 or any of examples B14-B19, wherein the inertial ordering channel comprises a spiral geometry.

[0133] Example B 17 includes the microfluidic chip of example B14 or any of examples B14-B19, wherein each channel of the plurality of flow channels has a width and a length configured to facilitate a controlled flow resistance.

[0134] Example B 18 includes the microfluidic chip of example B 14 or any of examples B14-B 19, wherein the outlet is configured without a droplet mixer to reduce a likelihood of blockage.

[0135] Example B19 includes the microfluidic chip of example B14 or any of examples B14-B18, wherein the flow-focusing channel junction is located at or proximate to a center of the microfluidic chip.

[0136] In some embodiments in accordance with the present technology (example B20), a system for temporal single-cell genomic sequencing includes: a microfluidic chip comprising a substrate having a plurality of How channels, a cell inlet, a microparticle inlet, a dispersion fluid inlet, a flow-focusing junction, and an outlet, wherein the plurality of flow channels comprises a cell flow channel coupled to the cell inlet, a microparticle flow channel coupled to the microparticle inlet, and a dispersion fluid flow channel coupled to dispersion fluid inlet; a cell reservoir in fluid communication with the cell inlet of the microfluidic chip and configured to contain a plurality of cells; a plurality of microparticle reservoirs, each microparticle reservoir configured to contain time-tagged microparticles having a unique time-tag barcode, wherein each of the microparticle reservoirs is in fluid communication with the microparticle inlet of the microfluidic chip; a plurality of timepoint valves, each timepoint valve associated with a respective microparticle reservoir and configured to control injection of time-tagged microparticles from the respective microparticle reservoir into the microfluidic chip; and a controller comprising a processor and a memory, the controller configured to coordinate sequential opening of the plurality of timepoint valves to enable temporal barcoding of cells at different time points through coencapsulation of the cells and selected time-tagged microparticles in the flow-focusing junction of the microfluidic chip.PCT Patent Application Docket No. 009062.8580.WQ00

[0137] Example B21 includes the system of example B20 or any of examples B20-B26, further comprising: a cell culture system configured to maintain cell viability in the cell reservoir, wherein the cell culture system comprises a water bath configured to maintain the cell reservoir at 37 °C.

[0138] Example B22 includes the system of example B21 or any of examples B20-B26, wherein the cell culture system further comprises a CO2 gas source configured to provide 5% CO2 to the cell reservoir.

[0139] Example B23 includes the system of example B20 or any of examples B20-B26, further comprising: a washing system in fluid communication with the microfluidic chip configured to clear fluid lines of residual microparticles or cells between successive injections.

[0140] Example B24 includes the system of example B20 or any of examples B20-B26, further comprising: a robotic system configured to move output containers to receive output collection samples from the outlet of the microfluidic chip in a selected output container based on a command from the controller.

[0141] Example B25 includes the system of example B20 or any of examples B20-B26, further comprising: a compressed air pressure controller; and a plurality of flow sensors, wherein each flow sensor is associated with a respective one of the microparticle flow channel, the cell flow channel, and the dispersion fluid flow channel, and wherein the compressed air pressure controller and the plurality of flow sensors are configured to enable high flow rates during a flushing phase and low flow rates during a droplet formation phase of operation of the system.

[0142] Example B26 includes the system of example B20 or any of examples B20-B25, further comprising: an automated vortex mixer associated with each of the plurality of microparticle reservoirs and configured to maintain microparticles evenly suspended before injection.

[0143] In some embodiments in accordance with the present technology (example B27), a method for single-cell temporal measurements includes sequentially introducing cells and time-tagged microparticles into a microfluidic chip to co-encapsulate the cells and the time-tagged microparticles at a plurality of time points, wherein the time-tagged microparticles introduced at each time point include a unique temporal barcode; and generating droplets containing the coencapsulated cells and time-tagged microparticles at each of the plurality of time points.

[0144] Example B28 includes the method of example B27 or any of examples B27-B44, further comprising: collecting the droplets from an outlet of the microfluidic chip; and processing the collected droplets to generate a sequencing library.PCT Patent Application Docket No. 009062.8580.WO00

[0145] Example B29 includes the method of example B28 or any of examples B27-B44, wherein the processing the droplets comprises: breaking the droplets; combining the time-tagged microparticles from the plurality of time points; and performing a reverse transcription reaction on the combined time-tagged microparticles.

[0146] Example B30 includes the method of example B27 or any of examples B27-B44, wherein the sequentially introducing the cells and the time-tagged microparticles into the microfluidic chip comprises controlling a plurality of valves to select which time-tagged microparticles are introduced at each time point.

[0147] Example B31 includes the method of example B27 or any of examples B27-B44, wherein the time-tagged microparticles comprise beads.

[0148] Example B32 includes the method of example B27 or any of examples B27-B44, wherein each of the time-tagged microparticles comprises a cell barcode and a unique molecular identifier.

[0149] Example B33 includes the method of example B32 or any of examples B27-B44, wherein each of the time-tagged microparticles further comprises a polynucleotide region configured to capture nucleic acid material released from cells.

[0150] Example B34 includes the method of example B33 or any of examples B27-B44, wherein the polynucleotide region comprises a PolyT region configured to capture mRNA.

[0151] Example B35 includes the method of example B27 or any of examples B27-B44, further comprising: lysing the cells to release nucleic acid material from the cells.

[0152] Example B36 includes the method of example B35 or any of examples B27-B44, wherein the lysing the cells occurs within the droplets in the microfluidic chip.

[0153] Example B37 includes the method of example B35 or any of examples B27-B44, wherein the lysing the cells occurs in at least one or both of a cell reservoir and a transport channel between the reservoir and the microfluidic chip.

[0154] Example B38 includes the method of example B35 or any of examples B27-B44, further comprising: capturing the released nucleic acid material on the time-tagged microparticles.

[0155] Example B39 includes the method of example B27 or any of examples B27-B44, wherein the plurality of time points comprises at least twelve time points, and wherein the method for single-cell temporal measurements is conducted in under two hours.

[0156] Example B40 includes the method of example B27 or any of examples B27-B44,PCT Patent Application Docket No. 009062.8580.WO00further comprising: performing a flushing operation between successive time points to clear residual cells or microparticles.

[0157] Example B41 includes the method of example B27 or any of examples B27-B44, further comprising: maintaining cell viability during an entirety of implementation of the method for at least a two hour period by maintaining a cell reservoir temperature at 37 °C in a presence of 5% CO2.

[0158] Example B42 includes the method of example B27 or any of examples B27-B44, wherein the generating the droplets comprises using inertial ordering to co-encapsulate one cell with one time-tagged microparticle in each droplet.

[0159] Example B43 includes the method of example B27 or any of examples B27-B44, wherein the generating droplets comprises introducing a dispersion fluid into the microfluidic chip to form the droplets.

[0160] Example B44 includes the method of example B43 or any of examples B27-B43, wherein the dispersion fluid comprises at least one of oil or water.Conclusion

[0161] Implementations of the subject matter and the functional operations described in this patent document can be implemented in various systems, digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. Implementations of the subject matter described in this specification can be implemented as one or more computer program products, i.e., one or more modules of computer program instructions encoded on a tangible and non-transitory computer readable medium for execution by. or to control the operation of, data processing apparatus. The computer readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, a composition of matter effecting a machine-readable propagated signal, or a combination of one or more of them. The term “data processing unit” or “data processing apparatus” encompasses all apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, or multiple processors or computers. The apparatus can include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them.PCT Patent Application Docket No. 009062.8580.WO00

[0162] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program does not necessarily correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g.. one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.

[0163] The processes and logic flows described in this specification can be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit).

[0164] Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. The essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices. Computer readable media suitable for storing computer program instructions and data include all forms of nonvolatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g.. EPROM, EEPROM, and flash memory devices. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0165] While this patent document contains many specifics, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain featuresPCT Patent Application Docket No. 009062.8580.WQ00that are described in this patent document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[0166] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Moreover, the separation of various system components in the embodiments described in this patent document should not be understood as requiring such separation in all embodiments.

[0167] Only a few implementations and examples are described and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.

Claims

1. PCT Patent Application Docket No. 009062.8580.WO00CLAIMSWhat is claimed is:

1. A method for temporal single-cell genomic sequencing, comprising:providing a microfluidic chip having a cell inlet, a bead inlet, and an outlet; injecting a first sample of cells from a cell reservoir into the cell inlet of the microfluidic chip;injecting a first set of time-tagged beads from a first bead reservoir of a plurality of bead reservoirs into the bead inlet of the microfluidic chip, wherein the first set of time-tagged beads comprises a first time barcode;co-encapsulating cells from the first sample of cells with time-tagged beads from the first set of time-tagged beads in droplets within the microfluidic chip to produce a first time-tagged sample;collecting the first time-tagged sample from the outlet of the microfluidic chip; injecting a second sample of cells from the cell reservoir into the cell inlet of the microfluidic chip;injecting a second set of time-tagged beads from a second bead reservoir of the plurality of bead reservoirs into the bead inlet of the microfluidic chip, wherein the second set of time-tagged beads comprises a second time barcode different from the first time barcode;co-encapsulating cells from the second sample of cells with time-tagged beads from the second set of time-tagged beads in droplets within the microfluidic chip to produce a second time-tagged sample; andcollecting the second time-tagged sample from the outlet of the microfluidic chip.

2. The method of claim 1, wherein the injecting the first set of time-tagged beads comprises opening a first timepoint valve associated with the first bead reservoir while maintaining a cell sample valve in an open position and maintaining a second timepoint valve associated with the second bead reservoir in a closed position.

3. The method of claim 1, further comprising:performing a flushing phase between collecting the first time-tagged sample and injectingPCT Patent Application Docket No. 009062.8580.WQ00the second sample of cells, wherein the flushing phase clears fluid lines of residual beads or cells from the first time-tagged sample.

4. The method of claim 1, wherein the injecting the second set of time-tagged beads occurs at an interval of seven minutes or less after the injecting the first set of time-tagged beads.

5. The method of claim 1, wherein each of the collecting the first time-tagged sample and the collecting the second time-tagged sample has a sampling duration of one minute or less.

6. The method of claim 1, wherein the co-encapsulating comprises using inertial ordering to co-encapsulate one bead with one cell in each droplet.

7. The method of claim 1, further comprising:injecting one or more additional sets of time-tagged beads from a corresponding one or more additional bead reservoirs of the plurality bead reservoirs into the bead inlet of the microfluidic chip at successive time intervals, wherein each additional set of time-tagged beads comprises a unique time barcode different from all other time barcodes.

8. The method of claim 7, wherein the method supports at least twelve unique time barcodes.

9. The method of claim 1, wherein the time-tagged beads comprise a cell barcode, a unique molecular identifier (UMI), a time-tag, and a polynucleotide region configured to capture mRNA released from lysed cells within the droplets.

10. The method of claim 1, further comprising:prior to the collecting, lysing the cells within the droplets to release mRNA from the cells; andcapturing the released mRNA on the time-tagged beads through molecular binding with a polynucleotide region on the time-tagged beads.

11. The method of claim 10, further comprising:breaking the droplets after collecting all time-tagged samples:combining beads from the first time-tagged sample and the second time-tagged sample;PCT Patent Application Docket No. 009062.8580.WQ00andperforming a reverse transcription reaction on the combined beads.

12. The method of claim 1, further comprising:maintaining the cell reservoir at 37°C and in a presence of 5% CO2 to maintain cell viability throughout a full course of the method.

13. The method of claim 1, further comprising:automatically moving outlet tubing to different collection tubes to prevent mixing of the first time-tagged sample with the second time-tagged sample.

14. A microfluidic chip for temporal single-cell genomic sequencing, comprising:a substrate having a plurality of flow channels including a cell flow channel, a microparticle flow channel, and a dispersion fluid flow channel;a cell inlet coupled to the cell flow channel configured to receive cells;a microparticle inlet coupled to the microparticle flow channel configured to receive time-tagged microparticles;a dispersion fluid inlet coupled to the dispersion fluid flow channel configured to receive at least one of oil or air;a flow-focusing channel junction in fluid communication with at least one region of the cell flow channel, the microparticle flow channel, and the dispersion fluid flow channel, wherein the flow-focusing junction is configured to receive a first fluid medium containing the cells, a second fluid medium containing the time-tagged microparticles, and the at least one of oil or air and to co-encapsulate cells with time-tagged microparticles in droplets,wherein the microparticle flow channel is configured as an inertial ordering channel comprising a spiral design to provide deterministic ordering of the time-tagged microparticles into the flow-focusing channel junction; andan outlet configured to output droplets containing co-encapsulated cells and time-tagged microparticles.

15. The microfluidic chip of claim 14, wherein the inertial ordering channel is configured to provide deterministic ordering of the microparticles to increase a number of droplets containing one microparticle and one cell.PCT Patent Application Docket No. 009062.8580.WO0016. The microfluidic chip of claim 14, wherein the inertial ordering channel comprises a spiral geometry.

17. The microfluidic chip of claim 14, wherein each channel of the plurality of flow channels has a width and a length configured to facilitate a controlled flow resistance.

18. The microfluidic chip of claim 14, wherein the outlet is configured without a droplet mixer to reduce a likelihood of blockage.

19. The microfluidic chip of claim 14, wherein the flow-focusing channel junction is located at or proximate to a center of the microfluidic chip.

20. A system for temporal single-cell genomic sequencing, comprising:a microfluidic chip comprising a substrate having a plurality of flow channels, a cell inlet, a microparticle inlet, a dispersion fluid inlet, a flow-focusing junction, and an outlet, wherein the plurality of flow channels comprises a cell flow channel coupled to the cell inlet, a microparticle How channel coupled to the microparticle inlet, and a dispersion fluid flow channel coupled to dispersion fluid inlet;a cell reservoir in fluid communication with the cell inlet of the microfluidic chip and configured to contain a plurality of cells;a plurality of microparticle reservoirs, each microparticle reservoir configured to contain time-tagged microparticles having a unique time-tag barcode, wherein each of the microparticle reservoirs is in fluid communication with the microparticle inlet of the microfluidic chip;a plurality of timepoint valves, each timepoint valve associated with a respective microparticle reservoir and configured to control injection of time-tagged microparticles from the respective microparticle reservoir into the microfluidic chip; anda controller comprising a processor and a memory, the controller configured to coordinate sequential opening of the plurality of timepoint valves to enable temporal barcoding of cells at different time points through co-encapsulation of the cells and selected time-tagged microparticles in the flow-focusing junction of the microfluidic chip.PCT Patent Application Docket No. 009062.8580.WQ0021. The system of claim 20, further comprising:a cell culture system configured to maintain cell viability in the cell reservoir, wherein the cell culture system comprises a water bath configured to maintain the cell reservoir at 37 °C.

22. The system of claim 21, wherein the cell culture system further comprises a CO2 gas source configured to provide 5% CO2 to the cell reservoir.

23. The system of claim 20. further comprising:a washing system in fluid communication with the microfluidic chip configured to clear fluid lines of residual microparticles or cells between successive injections.

24. The system of claim 20, further comprising:a robotic system configured to move output containers to receive output collection samples from the outlet of the microfluidic chip in a selected output container based on a command from the controller.

25. The system of claim 20, further comprising:a compressed air pressure controller; anda plurality of flow sensors, wherein each flow sensor is associated with a respective one of the microparticle flow channel, the cell flow channel, and the dispersion fluid flow channel, and wherein the compressed air pressure controller and the plurality of flow sensors are configured to enable high flow rates during a flushing phase and low flow rates during a droplet formation phase of operation of the system.

26. The system of claim 20. further comprising:an automated vortex mixer associated with each of the plurality of microparticle reservoirs and configured to maintain microparticles evenly suspended before injection.PCT Patent Application Docket No. 009062.8580.WQ0027. A method for single-cell temporal measurements, comprising:sequentially introducing cells and time-tagged microparticles into a microfluidic chip to co-encapsulate the cells and the time-tagged microparticles at a plurality of time points, wherein the time-tagged microparticles introduced at each time point include a unique temporal barcode; andgenerating droplets containing the co-encapsulated cells and time-tagged microparticles at each of the plurality of time points.

28. The method of claim 27, further comprising:collecting the droplets from an outlet of the microfluidic chip; andprocessing the collected droplets to generate a sequencing library.

29. The method of claim 28, wherein the processing the droplets comprises:breaking the droplets;combining the time-tagged microparticles from the plurality of time points; and performing a reverse transcription reaction on the combined time-tagged microparticles.

30. The method of claim 27, wherein the sequentially introducing the cells and the time-tagged microparticles into the microfluidic chip comprises controlling a plurality of valves to select which time-tagged microparticles are introduced at each time point.

31. The method of claim 27, wherein the time-tagged microparticles comprise beads.

32. The method of claim 27, wherein each of the time-tagged microparticles comprises a cell barcode and a unique molecular identifier.

33. The method of claim 32, wherein each of the time-tagged microparticles further comprises a polynucleotide region configured to capture nucleic acid material released from cells.

34. The method of claim 33, wherein the polynucleotide region comprises a PolyT region configured to capture mRNA.

35. The method of claim 27, further comprising:lysing the cells to release nucleic acid material from the cells.PCT Patent Application Docket No. 009062.8580.WQ0036. The method of claim 35, wherein the lysing the cells occurs within the droplets in the microfluidic chip.

37. The method of claim 35, wherein the lysing the cells occurs in at least one or both of a cell reservoir and a transport channel between the reservoir and the microfluidic chip.

38. The method of claim 35, further comprising:capturing the released nucleic acid material on the time-tagged microparticles.

39. The method of claim 27, wherein the plurality of time points comprises at least twelve time points, and wherein the method for single-cell temporal measurements is conducted in under two hours.

40. The method of claim 27, further comprising:performing a flushing operation between successive time points to clear residual cells or microparticles.

41. The method of claim 27, further comprising:maintaining cell viability during an entirety of implementation of the method for at least a two hour period by maintaining a cell reservoir temperature at 37°C in a presence of 5% CO2.

42. The method of claim 27, wherein the generating the droplets comprises using inertial ordering to co-encapsulate one cell with one time-tagged microparticle in each droplet.

43. The method of claim 27, wherein the generating droplets comprises introducing a dispersion fluid into the microfluidic chip to form the droplets.

44. The method of claim 43, wherein the dispersion fluid comprises at least one of oil or water.

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