System for processing cell samples, multi-microwell-array flow cell cartridge for use therein, and method of use thereof

Abstract

Systems for processing cellular samples are provided. System embodiments include a tray configured to receive a multi-microwell array flow cell cartridge and a collection magnet assembly configured to apply a uniform magnetic force to the flow cells of the cartridge when in an active position. Methods are also provided that may include introducing the multi-microwell array flow cell cartridge into the system, loading a sample into the multi-microwell array flow cell cartridge, actuating the collection magnet assembly to the active position to apply a uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to create a processed sample, and collecting the processed sample from the multi-microwell array flow cell cartridge. Additionally, multi-microwell array flow cell cartridges are disclosed. Embodiments of the cartridge include multiple fluid lanes, each including an inlet for receiving a liquid, a flow cell comprising a microwell array, and an outlet for discharging the liquid.

Description

[Background technology]

[0001] The ability to detect and quantify specific nucleic acid and protein molecules in individual cells is critical for understanding the role of cellular diversity in development, health and disease.

[0002] Flow cytometry has become a standard technique for high-throughput detection of protein markers on single cells and is widely adopted in basic research and clinical diagnostics. On the other hand, nucleic acid measurements, such as mRNA expression, are typically performed on bulk samples, obscuring the contributions from individual cells.

[0003] Methods and techniques such as probabilistic barcoding are useful for cell analysis. For example, probabilistic barcoding can be used to decipher the physiological condition of cells, for example, the protein and / or gene expression profile of a single cell, and determine their status using, for example, reverse transcription, polymerase chain reaction (PCR) amplification, and next-generation sequencing (NGS). However, the detected expression profile may be associated with two or more cells of different types, which may distort the interpretation of the expression profile.

[0004] Array technology has been used in biomedical research. Arrays comprise probes that can hybridize with target molecules bearing labels, e.g., fluorescence. Features on the array are small clusters of identical or similar probes bearing specific molecular sequences, e.g., DNA or RNA. By identifying the labeling pattern on the hybridized array, one can infer the hybridization occurring in the sample, which can further aid biomedical research. Loading stations for loading and recovering particles in array devices are described in U.S. Patent Nos. 5,629,999 and 5,729,999, the disclosures of which are incorporated herein by reference in their entireties.

[0005] To characterize the complexity of cellular systems, it is highly desirable to develop methods, devices, and systems for monitoring the expression of large numbers of genes across thousands of cells. Current technology enables the measurement of gene expression in single cells in massive parallel (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells as they colocalize with barcoded reagent beads within compartments. Summary of the Invention

[0006] The present inventors have recognized that there is a continuing need for improved systems and methods for preparing sequence-ready nucleic acid libraries from single cells. While the loading stations described in, for example, U.S. Patent Nos. 10,634,691 and 11,061,043 have improved the process for preparing cell samples, it has been discovered that further enhancements are desirable. Embodiments of the present invention fulfill these and other needs.

[0007] Aspects of the present invention include systems for processing cell samples. The system of the present invention includes a tray configured to receive a multi-microwell array flow cell cartridge and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the cartridge when in an active position. In certain cases, the retrieval magnet assembly is configured to apply the uniform magnetic force from a position above the tray. In some cases, the retrieval magnet assembly is configured to apply the uniform magnetic force from a position above the tray. In certain cases, the uniform magnetic force may be a magnetic field in the range of 650 Gauss to 1325 Gauss. In certain examples, the retrieval magnet assembly is configured to apply the uniform magnetic force via multiple magnets. In some such examples, the multiple magnets have alternating polarities. The number of magnets in the retrieval magnet assembly may vary, and in some cases, may range from 2 to 10 (e.g., 4). The type of magnet in the retrieval magnet assembly may also vary. In some cases, the magnets are rare earth magnets (e.g., neodymium magnets and / or samarium-cobalt magnets). The shape of the magnets may also vary. In some cases, the plurality of magnets are bar magnets. In other cases, the plurality of magnets are ring magnets. In some such cases, the plurality of magnets are arranged in a bull's-eye configuration. The systems of the present invention may be configured to adjust the position of the retrieval magnet assembly for processing a cell sample. In some embodiments, the retrieval magnet assembly is operable between an operative position, in which the retrieval magnet assembly is positioned adjacent to the tray, and a non-operative position, in which the retrieval magnet is positioned further away from the tray relative to the operative position.

[0008] In some versions, the system includes a sample collection container holder configured to receive multiple sample collection containers for collecting analytes from the cartridge. In some such versions, the sample collection container holder includes a counterweight configured to maintain the sample collection container in an upright position. The system and the sample collection container holder may also have complementary shapes so that the sample collection container holder can be received in the system in a single orientation. In some aspects, the system also includes multiple sample collection containers. The number of sample collection containers may vary and, in some cases, may range from 2 to 10 (e.g., 8). In particular examples, the system includes a number of sample collection containers that matches the number of flow cells in the cartridge. In some embodiments, the system includes a waste collection container for collecting liquid waste from the multi-microwell-array-flow cell cartridge. In some cases, the system includes an interlock configured to prevent collection of sample liquid into the waste collection container when the collection magnet assembly is in the activated position. In some cases, the tray includes a latch for holding the multi-microwell-array-flow cell cartridge. In selected versions, the system does not include a lysis magnet located below the tray.

[0009] In some cases, the system further includes a multi-microwell array flow cell cartridge. The subject multi-microwell array flow cell cartridges include multiple fluid lanes, each including an inlet for receiving liquid, a flow cell comprising a microwell array, and an outlet for discharging liquid. In certain cases, the multi-microwell array flow cell cartridge includes a number of fluid lanes ranging from 2 to 10 (e.g., 8). In some versions, each outlet is stepped to prevent siphoning of liquid from the flow cell. In some embodiments, each flow cell includes an elongated channel. The length of the elongated channel may vary, in some cases ranging from 50 mm to 100 mm. The number of microwells within each microwell array may also vary, in some cases ranging from 250,000 microwells to 300,000 microwells. The density of microwells within each microwell array may vary, in some cases ranging from 36,000 microwells / cm. 2 ~42,000 microwells / cm 2 may be in the range of

[0010] In some cases, the tray has a shape complementary to that of the multi-microwell-array-flow cell cartridge so that the multi-microwell-array-flow cell cartridge can be received in the tray in a single orientation. In some such cases, the multi-microwell-array-flow cell cartridge includes chamfered corners. In embodiments, the system further includes a drip receptacle positioned below the tray and configured to receive liquid discharged from the cartridge.

[0011] Aspects of the present invention also include methods for processing a cell sample. The subject methods include introducing a multi-microwell array flow cell cartridge (e.g., as described above) into a system of the present invention (e.g., as described above). The subject systems for the methods include a tray configured to receive the multi-microwell array flow cell cartridge and a collection magnet assembly configured to apply a uniform magnetic force to a flow cell of the multi-microwell array flow cell cartridge when in an active position. The method also includes loading a sample into the multi-microwell array flow cell cartridge, actuating the collection magnet assembly to the active position to apply a uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to create a processed sample, and collecting the processed sample from the multi-microwell array flow cell cartridge.

[0012] In certain cases, the method includes loading multiple different samples into different flow cells of the cartridge. The number of samples can vary, and in some cases can range from 2 to 10 (e.g., 8). Methods according to certain embodiments include loading the multi-well cartridge with a sample liquid followed by loading the multi-well cartridge with a lysis buffer. In some versions, the method includes loading the multi-well cartridge with a lysis buffer without using a lysis magnet. Methods according to selected versions of the invention include loading barcoded beads into the multi-well cartridge prior to actuation of the collection magnet assembly. In certain cases, the barcoded beads can include a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain. In selected embodiments, the target capture domain is a poly(T) sequence. In certain instances, the nucleic acid barcode further includes a unique molecular index (UMI).

[0013] The type of analysis performed in the method may vary as needed. In some cases, the method includes generating a sequence-ready nucleic acid library from the processed sample. In certain embodiments, the sequence-ready nucleic acid library is sequenceable by using a next-generation sequencing protocol. In further embodiments, the method is a method of genomic, epigenomic, transcriptomic, or proteomic analysis. In selected versions, the method is a method of multi-omic analysis, such as when the multi-omic analysis includes at least transcriptomic and proteomic analysis.

[0014] Aspects of the present invention also include multi-microwell array flow cell cartridges. The subject cartridges include a plurality of fluid lanes, each including an inlet for receiving a liquid, a flow cell comprising a microwell array, and an outlet for discharging the liquid. The disclosed multi-microwell array flow cell cartridges are configured for use with the subject systems and methods. Additionally, aspects of the present invention include sample collection container holders configured to receive a plurality of sample collection containers for collecting analytes from the multi-microwell array flow cell cartridges of the present invention. In some cases, the sample collection container holder has a shape complementary to a cellular analysis system such that the sample collection container holder can be received in the cellular analysis system in a single orientation.

[0015] Aspects of the present invention may further include kits. The kits of the present invention include the multi-microwell array flow cell cartridge of the present invention. As described above, the multi-microwell array flow cell cartridge of the present invention includes multiple fluid lanes, each of which includes an inlet for receiving a liquid, a flow cell including a microwell array, and an outlet for discharging the liquid. The kits may further include one or more devices for performing the methods of the present invention. For example, kits according to some embodiments include a sample collection container holder, one or more sample collection containers, and one or more waste collection containers. In some cases, the kits also include one or more reagents for performing the methods of the present invention. For example, kits according to some embodiments may include a hybridization buffer, a wash buffer, a reducing agent, and barcoded beads. [Brief explanation of the drawings]

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: [Figure 1A] 1A-1B illustrate a retrieval magnet assembly and tray according to certain embodiments, and actuation of the retrieval magnet assembly from a non-actuated position (FIGS. 1A-1B) to an actuated position (FIGS. 1C-1D). [Figure 1B] 1A-1B illustrate a retrieval magnet assembly and tray according to certain embodiments, and actuation of the retrieval magnet assembly from a non-actuated position (FIGS. 1A-1B) to an actuated position (FIGS. 1C-1D). [Figure 1C] 1A-1B illustrate a retrieval magnet assembly and tray according to certain embodiments, and actuation of the retrieval magnet assembly from a non-actuated position (FIGS. 1A-1B) to an actuated position (FIGS. 1C-1D). [Figure 1D]1A-1B illustrate a retrieval magnet assembly and tray according to certain embodiments, and actuation of the retrieval magnet assembly from a non-actuated position (FIGS. 1A-1B) to an actuated position (FIGS. 1C-1D). [Figure 1E] 1A-1B illustrate a retrieval magnet assembly and tray according to certain embodiments, and actuation of the retrieval magnet assembly from a non-actuated position (FIGS. 1A-1B) to an actuated position (FIGS. 1C-1D). [Figure 2A] 1 illustrates a retrieval magnet assembly according to certain embodiments. [Figure 2B] 1 illustrates a retrieval magnet assembly according to certain embodiments. [Figure 3A] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3B] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3C] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3D] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3E] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3F] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3G] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 3H] FIG. 1 illustrates a system for processing a cell sample according to certain embodiments. [Figure 4A] 1 illustrates a drip tray according to certain embodiments. [Figure 4B] 1 illustrates a drip tray according to certain embodiments. [Figure 5A] 10A-10C illustrate an interlock according to certain embodiments. [Figure 5B] 10A-10C illustrate an interlock according to certain embodiments. [Figure 6A]FIG. 1 illustrates a multi-microwell array flow cell cartridge according to certain embodiments. [Figure 6B] FIG. 1 illustrates a multi-microwell array flow cell cartridge according to certain embodiments. [Figure 6C] FIG. 1 illustrates a multi-microwell array flow cell cartridge according to certain embodiments. [Figure 7A] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7B] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7C] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7D] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7E] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7F] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7G] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 7H] 1A-1C are different views of a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 8] FIG. 1 illustrates alignment features of a multi-microwell-array-flow cell cartridge, according to certain embodiments. [Figure 9A] 1A-1C are different views of the microwells of the flow cell within the cartridge of the present invention. [Figure 9B-9C] 1A-1C are different views of the microwells of the flow cell within the cartridge of the present invention. [Figure 10]FIG. 1 illustrates fluid flow through a multi-microwell-array-flow cell cartridge according to certain embodiments. [Figure 11A] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11B] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11C] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11D] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11E] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11F] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11G] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 11H] 1 illustrates a cartridge carrier according to a specific embodiment of the present invention. [Figure 12A] 1A-1C illustrate a specimen collection container holder according to certain embodiments. [Figure 12B] 1A-1C illustrate a specimen collection container holder according to certain embodiments. [Figure 12C] 1A-1C illustrate a specimen collection container holder according to certain embodiments. [Figure 13] 1 is a flow diagram for carrying out the method of the present invention according to certain embodiments. [Figure 14A] 1 is a flow diagram for carrying out the method of the present invention according to certain embodiments. [Figure 14B] FIG. 1 is a schematic diagram showing a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), such as barcoded mRNAs or fragments thereof. [Figure 15A] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15B]FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15C] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15D] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15E] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15F] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15G] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15H] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15I] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 15J] FIG. 10 shows experimental data relating to the magnetic flux density of the magnet in the retrieval magnet assembly. [Figure 16A] FIG. 1 shows data demonstrating the high throughput of BD Rhapsody™ HT-Xpress. [Figure 16B] FIG. 1 shows data demonstrating the high throughput of BD Rhapsody™ HT-Xpress. [Figure 17A] FIG. 11 depicts data showing flexible cell throughput per lane with BD Rhapsody™ HT-Xpress. [Figure 17B] FIG. 11 depicts data showing flexible cell throughput per lane with BD Rhapsody™ HT-Xpress. [Figure 17C] FIG. 11 depicts data showing flexible cell throughput per lane with BD Rhapsody™ HT-Xpress. [Figure 17D]FIG. 11 depicts data showing flexible cell throughput per lane with BD Rhapsody™ HT-Xpress. [Figure 18A] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 18B] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 18C] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 19A] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 19B] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 19C] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 19D] FIG. 11 depicts data showing flexible throughput using a partial BD Rhapsody™ HT-Xpress 8-lane cartridge. [Figure 20A] FIG. 1 depicts data showing capture of cells of various sizes by BD Rhapsody™ HT-Xpress. [Figure 20B] FIG. 1 depicts data showing capture of cells of various sizes by BD Rhapsody™ HT-Xpress. [Figure 21A] FIG. 1 depicts data showing capture of fragile cells by BD Rhapsody™ HT-Xpress. [Figure 21B] FIG. 1 depicts data showing capture of fragile cells by BD Rhapsody™ HT-Xpress. [Figure 22A] FIG. 10 is a diagram of data for two-stage staining. [Figure 22B] FIG. 10 is a diagram of data for two-stage staining. [Figure 22C] FIG. 10 is a diagram of data for two-stage staining. [Figure 22D] FIG. 10 is a diagram of data for two-stage staining. [Figure 23A] FIG. 1 shows antigen-specific T cell profiling using BD Rhapsody™ Single-Cell Analysis. [Figure 23B] FIG. 1 shows antigen-specific T cell profiling using BD Rhapsody™ Single-Cell Analysis. [Figure 23C] FIG. 1 shows antigen-specific T cell profiling using BD Rhapsody™ Single-Cell Analysis, and includes, from top to bottom, SEQ ID NOs: 3-13. [Figures 24A-24B] FIG. 1 shows simultaneous profiling of mRNA, surface and intracellular proteins. [Figures 24C-24D] FIG. 1 shows simultaneous profiling of mRNA, surface and intracellular proteins. [Figures 24E-24L] FIG. 1 shows simultaneous profiling of mRNA, surface and intracellular proteins. [Figure 25A] FIG. 1 demonstrates the absence of batch effects between technical replicates. [Figures 25B-25C] FIG. 1 demonstrates the absence of batch effects between technical replicates. DETAILED DESCRIPTION OF THE INVENTION

[0017] A system for processing cellular samples is provided. The system, according to an embodiment of the present invention, includes a tray configured to receive a multi-microwell array flow cell cartridge and a collection magnet assembly configured to apply a uniform magnetic force to the flow cells of the cartridge when in an active position. A method of practicing the present invention is also provided, and embodiments include introducing a multi-microwell array flow cell cartridge into a system of the present invention, loading the multi-microwell array flow cell cartridge with a sample, actuating the collection magnet assembly to the active position to apply a uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to create a processed sample, and collecting the processed sample from the multi-microwell array flow cell cartridge. A multi-microwell array flow cell cartridge is also disclosed. The cartridge includes multiple fluid lanes, each of which includes an inlet for receiving a liquid, a flow cell comprising a microwell array, and an outlet for discharging the liquid.

[0018] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0019] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number preceded by the term, as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically stated number, the unstated near or approximately number may be a number that, in the context in which it is presented, represents the substantial equivalent of the specifically stated number.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

[0022] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0023] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0024] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be carried out in the order of events recited or in any other order which is logically possible.

[0025] Although the apparatus and methods are described for grammatical fluidity with functional descriptions, it is to be clearly understood that the claims should not be construed as necessarily limited by "means" or "step" limitation constructions unless expressly formulated under 35 U.S.C. § 112, but rather should be given the full range of meaning and equivalents of the definitions provided by the claims under the doctrine of equivalents, and that if a claim is expressly formulated under 35 U.S.C. § 112, then the full statutory equivalents under 35 U.S.C. § 112 should be given.

[0026] System for processing a cell sample As described above, aspects of the present invention include systems for processing cell samples. In some embodiments, the systems include structures or features designed to facilitate implementation of a workflow. In some embodiments, the workflow can include a series of steps for hybridizing nucleic acids from a plurality of cells with nucleic acids, e.g., barcoded nucleic acids, displayed on the surface of a plurality of barcode-containing beads. For example, in some embodiments, the workflow can include introducing a plurality of cells into a microwell array, introducing a plurality of barcoded nucleic acid-containing beads into the microwell array, lysing the plurality of cells such that nucleic acids, e.g., mRNA from the cells, hybridize with the barcoded nucleic acids associated with (e.g., present on) the surface of the plurality of beads, and then collecting the beads from the microwell array. In some cases, the workflow is a component of a next-generation sequencing (NGS) library preparation workflow.

[0027] As described above, the system of the present invention includes a tray configured to receive a multi-microwell array flow cell cartridge and a collection magnet assembly configured to apply a uniform magnetic force to the flow cells of the cartridge when in the operative position. By "uniform magnetic force" is meant that the magnetic force is applied uniformly across the area of ​​the multi-microwell array flow cell cartridge to which the magnetic force is applied. In other words, the magnetic force applied to one portion of the multi-microwell array flow cell cartridge has minimal difference, such as 1% or less, such as 20% or less, such as 15% or less, such as 10% or less, or 5% or less, compared to the magnetic force applied to another portion of the multi-microwell array flow cell cartridge. In embodiments, the uniform magnetic force of the present invention results in an amount of magnetic force comparable to (including the same as) that applied to each flow cell of a multi-microwell array flow cell cartridge when the cartridge is placed in a system, such as when the amount of magnetic force applied to any two given flow cells differs by no more than 20%, such as no more than 15%, such as no more than 10%, such as no more than 5%, including no more than 1%. The uniform magnetic force may be of various strengths. For example, in some cases, the uniform magnetic force is a magnetic field in the range of 650 Gauss to 1,325 Gauss, including 650 Gauss to 700 Gauss, such as 700 Gauss to 750 Gauss, such as 750 Gauss to 800 Gauss, such as 800 Gauss to 850 Gauss, such as 850 Gauss to 900 Gauss, such as 900 Gauss to 950 Gauss, such as 950 Gauss to 1,000 Gauss, such as 1,000 Gauss to 1,050 Gauss, such as 1,050 Gauss to 1,200 Gauss, such as 1,200 Gauss to 1,250 Gauss, such as 1,250 Gauss to 1,300 Gauss, and 1,300 Gauss to 1,325 Gauss.

[0028] In embodiments, a tray is disposed on the top surface of the system body. In some embodiments, the tray is capable of accepting a multi-microwell array flow cell cartridge within the system. In some embodiments, the tray can be sized, positioned, or otherwise configured to orient the multi-microwell array flow cell cartridge to a predetermined location within the system. The predetermined location can facilitate interaction of the multi-microwell array flow cell cartridge with other features of the filling station. In some embodiments, the tray can releasably secure the cartridge within the system. For example, in some embodiments, the tray has a shape complementary to the shape of the multi-microwell array flow cell cartridge so that the multi-microwell array flow cell cartridge can be accepted into the tray in a single orientation. For example, in some cases where the multi-microwell array flow cell cartridge includes one or more chamfered corners (described in more detail below), the tray is configured to accept a cartridge having a corresponding shape, i.e., a chamfered corner. In this manner, the chamfered corner may be used as an orientation feature for cartridge placement.

[0029] In certain cases, the system includes one or more retention mechanisms configured to maintain the multi-microwell array flow cell cartridge in place when the cartridge is placed in the tray. In some such cases, the tray includes a latch for retaining the multi-microwell array flow cell cartridge. In selected embodiments, the latch is spring-loaded. The spring-loaded latch may be pushed back when the cartridge is inserted, applying a force to the cartridge that restrains the cartridge in place. In some cases, the latch includes one or more protruding features that allow a user to slide the latch back when removing the cartridge. The latch may be constructed using any convenient technique. In some embodiments, the latch is injection molded. In some such embodiments, the latch comprises an injection-moldable polymer. Any convenient injection-moldable polymer may be employed. Injection-moldable polymers may include, but are not limited to, acrylonitrile butadiene styrene (ABS), polycarbonate (PC), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polypropylene (PP), polybutylene terephthalate (PBT), polyphenylsulfone (PPSU), polyetheretherketone (PEEK), and polyetherimide (PEI). If the latch is spring-loaded, the spring may optionally be made of stainless steel (e.g., 18-8 stainless steel) to protect against oxidation by nearby liquids. In some cases, the latch further includes a latch plate configured to secure the latch to the system. For example, in some cases, the latch plate is configured to secure the latch to the system via one or more screws. If present, the latch plate may be constructed of any suitable material. In some cases, the latch plate is constructed from a metallic material, including, but not limited to, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In an embodiment, the latch plate is made from aluminum, such as 6061 aluminum.

[0030] In embodiments, the tray also includes a cartridge catch. The cartridge catch may be positioned on the opposite side of the tray from the latch and may exert a downward force on the cartridge when the cartridge is placed in the tray. The downward force may be sufficient to prevent the cartridge from being removed from the tray until the latch is disengaged. The cartridge catch may be secured to the system in any convenient manner, such as with one or more screws. If present, the cartridge catch may be constructed from any suitable material. In some cases, the cartridge catch is constructed from a metallic material, including, but not limited to, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In embodiments, the cartridge catch is made from aluminum, such as 6061 aluminum.

[0031] The subject system also includes a retrieval magnet assembly. As described above, the retrieval magnet assembly is configured to apply a uniform magnetic force to the flow cells of the cartridge when in the operative position. The retrieval magnet assembly may be employed, for example, when it is desirable to retrieve beads in the microwells of the cartridge. The retrieval magnet assembly is switchable between an operative position and a non-operative position. The operative position is a position in which the retrieval magnet assembly applies a magnetic force to the flow cells of the cartridge, while the non-operative position is a position in which the retrieval magnet assembly applies a smaller magnetic force (including no magnetic force) to the flow cells of the cartridge compared to the operative position. In the non-operative position, the retrieval magnet assembly is positioned further away from the multi-microwell-array-flow cell cartridge in at least one direction compared to the operative position. In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force to the multi-microwell-array-flow cell cartridge from a position above the tray. In some embodiments, when in the operative position, the lower surface of the retrieval magnet assembly can be parallel to the upper surface of the multi-microwell-array-flow cell cartridge. In some cases, the lower surface of the retrieval magnet assembly may be 1.0 mm or about 1.0 mm away from the upper surface of the multi-microwell array flow cell cartridge when in the activated position. In selected examples, the lower surface of the retrieval magnet assembly may be 0.5 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, less than 0.5 mm, less than 1.0 mm, 1.0 mm or less, less than 1.5 mm, less than 2.0 mm, less than 2.5 mm, less than 3.0 mm, or between 0.5 mm and 1.5 mm away from the upper surface of the multi-microwell array flow cell cartridge. In some embodiments, the system can include a retrieval magnet assembly actuator. In some embodiments, the actuator can be actuated to transition the retrieval magnet assembly between the inactivated position and the activated position.

[0032] While two positions for the retrieval magnet assembly are described above, it should be appreciated that in certain embodiments, the retrieval magnet assembly may be moved through three or more positions. In some embodiments, the retrieval magnet assembly may be moved between discrete positions. In some embodiments, the retrieval magnet assembly may be moved through a continuous range of positions. In embodiments, the positions of the retrieval magnet assembly allow for the application of varying amounts of magnetic force to magnetic particles in a multi-microwell-array-flow cell cartridge when the cartridge is placed in a tray.

[0033] In some embodiments, the system does not include a lysis magnet below the tray. A "lysis magnet" is a magnet used during the lysis step in a cell sample preparation method. For example, a lysis magnet may attract and retain beads into the microwells while a lysis buffer is pipetted through the flow cell. For example, the magnet 120 disclosed in U.S. Pat. Nos. 10,634,691 and 11,061,043 is considered herein to be a lysis magnet that is not present in embodiments of the system. Prior to the filing of the present disclosure, it was believed that a lysis magnet was necessary to prevent beads from being washed away. However, the inventors surprisingly realized that the lysis magnet could be removed from the design, and the beads would remain in the microwells during application of the lysis buffer.

[0034] In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force through multiple magnets. In such cases, the number of magnets in the multiple magnets may vary and may range from 2 to 10, such as from 2 to 6, and including from 3 to 5. For example, the number of magnets may be 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the retrieval magnet assembly includes four magnets. The magnets in the retrieval magnet assembly may include any suitable magnet. The magnets of interest include, but are not limited to, neodymium magnets, rare earth magnets such as samarium-cobalt magnets, and combinations thereof. The size and shape of the magnets may also vary. In some cases, the magnets are bar magnets. When the magnets are bar magnets, the length of each bar magnet may vary and in some cases may range from 50 mm to 500 mm, such as from 60 mm to 200 mm, such as from 70 mm to 80 mm, and including from 75 mm to 125 mm. In some cases, the bar magnet has a length of 76.2 mm. The width of the bar magnet may also vary, in some cases ranging from 5 mm to 20 mm, such as from 6 mm to 18 mm, and including from 10 mm to 15 mm. In some cases, the magnet has a width of 12.7 mm. The thickness of the magnet may also vary, in some cases ranging from 0.5 mm to 10 mm, such as from 1 mm to 5 mm, and including from 2 mm to 4 mm. In some cases, the width of the magnet is 3.175 mm. In other embodiments, the magnet is a ring magnet. In some such embodiments, the ring magnets are arranged concentrically, for example, in a bull's eye configuration. The magnets may be the same shape or different shapes. In some embodiments, the magnets have different shapes and / or sizes.

[0035] The placement of magnets relative to one another within a retrieval magnet assembly may vary. In some cases, the distance between adjacent magnets within a plurality of magnets ranges from 0.1 mm to 20 mm, such as from 1 mm to 15 mm, and including from 1 mm to 9 mm. In some cases, adjacent magnets may be separated by 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, or 12 mm, or any intermediate non-integer value between any two of these values. The distance separating adjacent magnets may be the same or different. In some cases, each magnet within a retrieval magnet assembly is separated from adjacent magnets by the same distance. In other cases, at least one magnet is separated from an adjacent magnet by a distance different from the distance separating another pair of adjacent magnets. In one example involving four bar magnets, the first and second magnets are separated by 2 mm, the second and third magnets are separated by 8 mm, and the third and fourth magnets are separated by 2 mm.

[0036] In embodiments, the plurality of magnets have alternating polarities. Without being bound by theory, it is believed that alternating the polarities of the magnets allows magnetic field lines coming from the north pole of an adjacent magnet to pass through the cartridge before entering the south pole. For example, in some embodiments, adjacent magnets in a retrieval magnet assembly are arranged in an N, S, N, S, ... configuration, where "N" points north and "S" points south, and the pattern can be repeated as many times as there are magnets in the plurality of magnets. In an alternative embodiment, the magnets in a retrieval magnet assembly are arranged in an S, N, S, N, ... configuration.

[0037] 1A-1E illustrate a retrieval magnet assembly and tray according to certain embodiments of the present invention. FIGS. 1A and 1B show different views of system 100 with retrieval magnet assembly 101 in an inactive position. Retrieval magnet assembly 101 is positioned away from tray 102. FIGS. 1C and 1D show different views of system 100 with retrieval magnet assembly 101 in an active position where retrieval magnet assembly 101 is positioned adjacent to tray 102. FIG. 1E is another view of system 100 showing latch 104, multi-microwell-array-flow cell cartridge 106, latch plate 105, and cartridge catch 103. Latch 104 and cartridge catch 103 are configured to maintain multi-microwell-array-flow cell cartridge 106 in tray 102. Latch 104 is spring-loaded and can be pushed back to remove multi-microwell-array-flow cell cartridge 106.

[0038] 2A-2B show different views of a retrieval magnet assembly according to certain embodiments. FIG. 2A shows retrieval magnet assembly 201 in an activated position. Retrieval magnet assembly 201 is positioned in an upper position relative to tray 202. FIG. 2B shows multiple magnets within retrieval magnet assembly 201. Magnets 203a-203d are bar magnets with alternating polarities and arranged in a N, S, N, S configuration.

[0039] In some embodiments, the system also includes a sample collection container holder configured to receive multiple sample collection containers for collecting analytes from the cartridge. The sample collection container holder is configured to be received within the system at a lower position than the tray and, if present, the cartridge, and to position the sample collection containers so that liquid from the cartridge can be received in the sample collection containers. In selected embodiments, the sample collection container holder includes a counterweight configured to maintain the sample collection container holder in an upright position. In other words, the sample collection container holder does not easily tip over when removed from the system. In some cases, the counterweight may be positioned at a lower position than the position at which the sample collection containers are received. In some cases, the system (e.g., a drawer of the system) and the sample collection container holder have complementary shapes so that the sample collection container holder can be received within the system in a single orientation. In other words, the sample collection container holder has a poka-yoke design. Further details regarding the sample collection container holders of the present invention are provided below.

[0040] In some embodiments, the system further includes a plurality of sample collection containers within the sample collection container holder. The sample collection containers may be any suitable liquid container. Suitable sample collection containers include, but are not limited to, test tubes, conical tubes, multi-compartment containers such as microtiter plates (e.g., 96-well plates), centrifuge tubes, culture tubes, microtubes, caps, cuvettes, bottles, linear polymer containers and bags, other types of containers, and the like. In some cases, the sample collection containers are test tubes. In certain cases, the opening to each collection container may include a cover, such as a valve, that can be reversibly closed as desired. The sample collection container holder may accommodate any suitable number of sample collection containers, for example, the number of sample collection containers ranges from 2 to 10, including 7 to 9. In some cases, the system includes eight sample collection containers.

[0041] In some cases, the system also includes a waste collection container for collecting liquid waste from the multi-microwell array flow cell cartridge, which may include, but is not limited to, conical tubes, centrifuge tubes, culture tubes, bottles, linear polymer containers, bags, other types of containers, and the like.

[0042] In some embodiments, the system of the present invention includes a drawer configured to hold one or more items, the drawer being movable between a plurality of different positions. In certain cases, the drawer is configured to receive at least one of a sample collection container holder and a waste collection container. In some such examples, the drawer is configured to receive both a sample collection container holder and a waste collection container. In some embodiments, the drawer is movable to at least one position where at least one (including all) of the sample collection containers in the sample collection container holder and / or the waste collection container align with one or more outlets of the cartridge. In some embodiments, the system includes a drawer actuator, the drawer actuator being movable between a plurality of different positions, and movement of at least some of the drawer actuator causes movement of the drawer. In some embodiments, the drawer may be movable along a guide rail. In some embodiments, the guide rail is positioned to engage a top surface of the drawer, a side surface of the drawer, or a bottom surface of the drawer. In some embodiments, the guide rail is positioned to reduce contact with spills or splashes of liquid.

[0043] The system may also include a drip tray designed to cover the linear rail and catch liquid when the cartridge is not over a waste or sample collection container. In certain cases, the drip tray is removable (e.g., for cleaning). In some cases, the drip tray has high boundary walls to ensure liquid remains in the tray and an ergonomic handle for removal. In certain cases, the drip tray is movable along the guide rail and actuable by a drawer actuator.

[0044] 3A-3H show different views of a system for processing a cellular sample in accordance with certain embodiments of the present invention. FIG. 3A shows a front view of system 300, FIG. 3B shows a rear view of system 300, FIG. 3C shows a top view of system 300, FIG. 3D shows a bottom view of system 300, FIGS. 3E-3F show views of system 300 at a 45° angle, and FIGS. 3G-3H show side views of system 300. System 300 includes a retrieval magnet assembly 301, a tray 302, a retrieval magnet assembly actuator 303, a latch 304, a sample collection container holder 305, a waste collection container 306, a drawer actuator 307, a drawer 308, and a drip tray 309. Retrieval magnet assembly 301 is actuable into an active position via retrieval magnet assembly actuator 303. When in the active position, retrieval magnet assembly 301 is configured to apply a uniform magnetic force to the multi-microwell-array-flow cell cartridge when the multi-microwell-array-flow cell cartridge is positioned in tray 302. System 300 further includes a latch 304 configured to maintain the multi-microwell-array-flow cell cartridge in tray 302. In this embodiment, latch 304 is a spring-loaded latch configured to be pushed rearward when the multi-microwell-array-flow cell cartridge is loaded into tray 302, exerting a force on the cartridge and thereby maintaining its position.

[0045] 3A-3H, drawer 308 includes sample collection container holder 305 and waste collection container 306. Drawer 308 has recesses complementary to the shapes of sample collection container holder 305 and waste collection container 306 so that sample collection container holder 305 and waste collection container 306 fit within drawer 308 in only a single orientation. Drawer 308 is operably connected to drawer actuator 307 configured to move drawer 308 between different positions within system 300. For example, drawer 308 can be moved to a first position (shown in FIGS. 3A-3H) in which the drawer is positioned outside of system 300. In the first position, waste collection containers, sample collection containers, and / or sample collection container holders may be added, removed, or replaced as needed. Using the drawer actuator 307, the drawer 308 can be moved to a sample collection position where liquid can be received into one or more sample collection containers in the sample collection container holder 305, or to a waste recovery position where liquid can be received into the waste collection container 306. The drawer 308 can also be moved via the drawer actuator 307 to one or more neutral positions where the drawer is positioned within the system 300 but is not positioned to receive liquid. Figure 3F shows a drip tray 309 configured to collect liquid that is not received into either the sample collection containers or the waste collection containers.

[0046] 4A-4B illustrate the drip tray and how the drip tray and drawer move through the system of the present invention. FIG. 4A shows the drip tray 400 including an ergonomic handle 402 and a high boundary wall 401 to facilitate removal and / or handling of the drip tray 400 so that liquid remains in the tray. FIG. 4B illustrates the position of the drip tray 400 relative to other components of the system, according to certain embodiments. As shown in FIG. 4B, the tray 400 is operatively connected to the drawer actuator 407 such that movement of the drawer actuator 407 moves both the tray 400 and the drawer 408, along with the specimen collection container holder 405 and waste collection container 406, if present, through the system along guide rails 410.

[0047] Aspects of the system further include an interlock configured to prevent collection of sample liquid into a waste collection container when the retrieval magnet assembly is in the activated position. As described above, when it is desired to obtain sample liquid from the cartridge (e.g., into one or more sample collection containers), the retrieval magnet assembly may be activated (e.g., by a user) to the activated position. The inventors have discovered that a common error among users of conventional systems is that the sample is retrieved into a waste container rather than a sample collection tube. The interlock described herein is configured to prevent accidental retrieval by locking the drawer in place during a retrieval operation. In other words, the drawer cannot be moved when the retrieval magnet is in the activated position because the interlock restricts its movement. In some embodiments, when a sample collection container is present in the sample collection container holder, the drawer is restricted to a position such that the sample collection container is positioned below the cartridge outlet, thereby allowing sample liquid to be received by the sample collection container. In some cases, the retrieval magnet assembly is operably connected to the interlock such that movement (i.e., activation) of the retrieval magnet assembly also causes movement of the interlock. The interlock engages the drawer and prevents movement of the drawer (e.g., along the guide rails), and the return of the retrieval magnet assembly to its inactive position disengages the interlock from the drawer so that the drawer can move freely when actuated by the drawer actuator.

[0048] 5A-5B illustrate an interlock according to certain embodiments. The system 500 includes a retrieval magnet assembly 501, a retrieval magnet assembly actuator 503, a drawer actuator 507, a drawer 508, and an interlock 511 operably connected to the retrieval magnet assembly 501. FIG. 5A illustrates the retrieval magnet assembly 501 in an inactive position. The interlock 511 is not engaged with the drawer 508, allowing the drawer to move freely via the drawer actuator 507. FIG. 5B illustrates the retrieval magnet assembly 501 in an active position. The interlock 511 is engaged with the drawer 508, resulting in restricted movement of the drawer 508.

[0049] Multi-microwell array flow cell cartridge As noted above, embodiments of the present invention further include multi-microwell array flow cell cartridges. A subject multi-microwell array flow cell cartridge (also referred to herein as a "cartridge") includes multiple fluid lanes, each of which includes an inlet for receiving fluid, a flow cell comprising a microwell array, and an outlet for discharging fluid. A "multi-microwell array flow cell" refers to a cartridge having multiple (i.e., multiple) flow cells, each of which includes a microwell array. Each flow cell is a component of a fluid lane, i.e., a path through which fluid can pass from an inlet to an outlet. The microwell array of the cartridge, when used for cell capture, can result in a cell capture rate of 50% or greater, including 55% or greater, such as 60% or greater, such as 65% or greater, such as 70% or greater, such as 75% or greater, such as 80% or greater, such as 85% or greater, such as 90% or greater, and including 95% or greater. In some cases, the cartridges of the present invention can be reused. For example, if one lane of a cartridge is used for a particular assay, the remaining lanes of the cartridge may be used for the same or different assays at one or more different times. In some cases, a single multi-microwell-array-flow cell cartridge may be used one or more times, including two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, and eight or more times. In some cases, a partially used cartridge (i.e., a cartridge in which one or more lanes have been used for an assay) is stable (i.e., remains operational) for one month or more, including two or more months or more, three or more months or more, such as four or more months, five or more months, and six or more months. In some embodiments, the use of a multi-microwell-array-flow cell cartridge minimizes batch effects between fluid lanes, including in cases where batch effects are negligible.

[0050] The number of fluid lanes in a cartridge may vary. In certain cases, a multi-microwell array flow cell cartridge comprises a number of fluid lanes ranging from 2 to 20, such as 2 to 15, including 2 to 10. For example, in embodiments, a cartridge comprises two lanes, three lanes, four lanes, five lanes, six lanes, seven lanes, eight lanes, nine lanes, or ten lanes. In certain embodiments, a multi-microwell array flow cell cartridge comprises eight fluid lanes. Each fluid lane comprises a flow cell comprising a microwell array. Further details regarding microwell arrays and flow cells are provided below. In some cases, a cartridge comprises multiple subsets of flow cells. In other words, multiple fluid lanes may be composed of smaller sets of fluid lanes that can be manufactured separately. Each subset may comprise a number of flow cells ranging from 2 to 6, such as 3 to 5. In some cases, each subset comprises four flow cells.

[0051] The inlets of each fluid lane may vary. In some cases, the inlets are configured with gaskets. In some cases, the inlets include gaskets configured to taper the side locks of pipette tips (e.g., when the pipette tip is introduced into the inlet so that liquid can be introduced into the fluid channel). The gaskets may be configured such that the taper locks have an overall Z-axis tolerance in the range of 0.1 mm to 1 mm, such as 0.2 mm to 0.8 mm, such as 0.3 mm to 0.7 mm, and including 0.4 mm to 0.6 mm. In some cases, the taper locks have an overall Z-axis tolerance of 0.5 mm. In some cases, the Z-axis tolerance is sufficient to account for variations in pipette tip height across different pipette tips. The hardness of the gaskets may vary. In some cases, the gaskets have a Shore durometer in the range of 1 to 100, such as 5 to 90, and including 10 to 80. The inlet may be constructed from any suitable material, including, but not limited to, silicon, fused silica, glass, any of a variety of polymers, e.g., polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as Teflon (PTFE), metals (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof.

[0052] The outlets of each fluid lane may similarly vary. In some cases, the outlets are configured to induce droplet formation. In some such cases, the outlets are configured to induce droplet formation over a range of fluid flow rates from 5 μL / s to 1,000 μL / s, including from 10 μL / s to 700 μL / s, such as from 15 μL / s to 600 μL / s, and including from 20 μL / s to 500 μL / s. In selected embodiments, the outlets are tapered conical orifices. The orifices are designed to accommodate a wide variety of fluids with different surface energies and maintain uniform droplet formation of the fluids by having specific inner and outer diameters, taper angles, and lengths. In some cases, the outlets have inner diameters ranging from 0.550 mm to 0.650 mm, including from 0.500 mm to 1.200 mm and from 0.750 mm to 1.050 mm. In some cases, the outlet has an outer diameter in the range of 1.000 mm to 2.500 mm, such as 1.200 mm to 1.300 mm, and including 1.400 mm to 1.600 mm. In some versions, the outlet has a taper angle in the range of 2° to 20°, such as 3° to 5°, such as 9° to 11°, and including 4° to 6°. In some cases, the outlet has a length in the range of 2.500 mm to 5.500 mm, such as 2.750 mm to 3.250 mm, and including 3.900 mm to 4.100 mm. The outlet may be constructed from any suitable material, including, but not limited to, silicon, fused silica, glass, any of a variety of polymers, e.g., polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as Teflon (PTFE), metal (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof.

[0053] In some cases, the outlet is a stepped outlet. "Stepped" means that the outlet comprises a raised portion that the fluid must travel before being discharged. In selected embodiments, the step in the outlet is sufficient to prevent siphoning of the liquid from the flow cell. In some cases, the outlet is configured with a siphon lid. In such cases, the siphon lid is configured to create a channel along the raised portion of the stepped outlet so that the fluid is directed toward and discharged from the outlet.

[0054] The inlet and outlet features of the cartridge can be designed to provide a convenient, leak-proof fluid connection with the instrument or can serve as open reservoirs for manual pipetting of samples and reagents into and out of the cartridge. Examples of convenient mechanical designs for the inlet and outlet port connectors can include, but are not limited to, threaded connectors, Luer lock connectors, Luer slip or "slip tip" connectors, press-fit connectors, and the like. The inlet and outlet ports of the cartridge can further comprise caps, spring-loaded covers or closures, or polymeric membranes that are opened or punctured when the cartridge is placed in the instrument to help prevent contamination of the cartridge's interior surfaces during storage or to prevent fluid spillage when the cartridge is removed from the instrument. The outlet port of the cartridge can further comprise a removable sample collection chamber suitable for interfacing with a stand-alone PCR thermal cycler or sequencing instrument.

[0055] In some embodiments, the inlets and outlets can direct fluid flow through the fluidic channels, thereby contacting the microwells with the fluid. In some embodiments, the device includes a pipette tip interface for loading or removing a cell sample, an assay reagent, a bead suspension, waste from the device, or a combination thereof. The device can contain a cell sample, an assay reagent, a bead suspension, or a combination thereof.

[0056] The cartridges of the present invention may further include a housing. The subject housing is configured to accommodate cartridge components such as the flow cell, inlet, and outlet. The housing may comprise any convenient material, including, but not limited to, silicon, fused silica, glass, any of a variety of polymers, such as polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as Teflon (PTFE), metals (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof. In some cases, an outlet is provided within the housing.

[0057] Figures 6A-6C illustrate one embodiment of the claimed cartridge. Figure 6A shows an exploded view of the cartridge, including a flow cell 601, gasket 602, and housing 603. It also shows a siphon lid 604 configured to be positioned adjacent to the outlet within housing 603 to create a channel over the raised portion of the outlet. Figure 6B shows an alternative view of gasket 602. As shown in Figure 6B, gasket 602 creates a taper lock around pipette tip 605 so that liquid can fill the fluid lanes of the cartridge. Figure 6C shows an outlet 607 with a tapered conical orifice.

[0058] Figures 7A-7H show various views of a multi-microwell array flow cell cartridge according to certain embodiments. Figures 7A-7B show a side cross-sectional view of multi-microwell array flow cell cartridge 700. Figure 7C shows a top view of cartridge 700. Cartridge 700 shows inlets 701a-701h, flow cells 702a-702h, and outlets 703a-703h. Figure 7D shows a bottom view of cartridge 700. Figure 7E shows a view from the top of cartridge 700 at a 45° angle, and Figure 7F shows a view from the bottom of cartridge 700 at a 45° angle. Figures 7G and 7H show side cross-sectional views of cartridge 700.

[0059] In some cases, the cartridge includes one or more alignment features configured to facilitate assembly of the cartridge and positioning of the cartridge within the tray of the system of the invention. For example, in some embodiments, the cartridge of the invention includes a chamfered corner (i.e., a transition edge between two edges of the cartridge). The chamfered corner may optionally be characterized by a specific angle relative to a straight edge of the cartridge. In some such cases, the angle may be in the range of 30° to 60°, such as 35° to 55°, such as 40° to 50°, and including 44° to 46°. As described above, the tray may be configured so that the cartridge can only be placed within the tray in a single orientation. In other words, the tray and cartridge have corresponding shapes. The cartridge may further include an aligner so that the flow cell can only be attached to the housing in a single orientation. The aligner may optionally take the form of a protrusion within the cartridge housing. In some embodiments, the tray and cartridge comply with ANSI / SLAS microplate standards. These standards can be accessed at https: / / www(dot)slas(dot)org / education / ansi-slas-microplate-standards / .

[0060] 8 shows a bottom view of a multi-microwell array flow cell cartridge according to certain embodiments of the invention, illustrating its associated alignment features. Cartridge 800 includes chamfered corners 801 configured to orient cartridge 800 within a tray (not shown) of a system of the invention. Cartridge 800 also includes aligners 802 and 803 configured to align a set of flow cells 804 upon installation.

[0061] Flow cell Each flow cell design can include multiple microarray chambers interfacing with multiple microwell arrays so that one or more different cell samples can be processed in parallel. The flow cell design can further include features for creating a consistent (e.g., uniform) flow velocity profile across the width of the array chamber, i.e., "plug flow," to provide efficient (e.g., uniform) delivery of cells and beads to the microwells, for example, by using a porous barrier positioned near the chamber inlet and upstream of the microwell array as a "flow diffuser," or by dividing each array chamber into several subsections that collectively cover the same total array area but through which divided inlet fluid streams flow in parallel. In some embodiments, the flow cell can encapsulate or incorporate two or more microwell array substrates.

[0062] In general, the dimensions of the fluid channel and array chamber in a flow cell design are optimized to (i) provide efficient (e.g., uniform) delivery of cells and beads to the microwell array and (ii) minimize sample and reagent consumption. In embodiments, each flow cell comprises an elongated channel. In some cases, the length of the elongated channel may be characterized by a straight line, i.e., the channel has no curves. The length of the elongated channel may vary. In certain cases, the length ranges from 20 mm to 500 mm, including 25 mm to 400 mm, such as 30 mm to 300 mm, such as 40 mm to 300 mm, such as 45 mm to 200 mm, and 50 mm to 100 mm. The width of the elongated channel may vary in different implementations, for example, from 0.1 mm to 100 mm. In some embodiments, the width can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mm, or a number or range between or about any two of these values. In some embodiments, the width can be at least or at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mm.

[0063] The height of the fluidic channels can vary in different implementations, for example, in the range of 0.1 mm to 100 mm, in some embodiments, the height can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm, or a number or range of heights between any two of these values, or can be about these values. In some embodiments, the height can be at least or at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm.

[0064] The flow cell can be fabricated using a variety of techniques and materials known to those skilled in the art. Typically, the flow cell is fabricated as a separate component and then mechanically clamped or permanently bonded to the microwell array substrate. Examples of suitable fabrication techniques include conventional machining, CNC machining, injection molding, 3D printing, laser or die-cutting, alignment and lamination of one or more layers of polymer film, or any of several microfabrication techniques such as photolithography and wet chemical etching, dry etching, deep reactive ion etching, or laser micromachining.

[0065] Once the flow cell components are fabricated, they can be mechanically attached to the microwell array substrate, for example, by clamping them against the microwell array substrate (with or without a gasket), or they can be directly bonded to the microwell array substrate using any of a variety of techniques known to those skilled in the art (depending on the choice of materials used), such as anodic bonding, thermal bonding, or any of a variety of adhesives or adhesive films, including epoxy-, acrylic-, silicone-, UV-curable, polyurethane-, or cyanoacrylate-based adhesives. In some embodiments, the substrate can form the fluidic channel bottom of the fluidic channel, or the substrate can be on the fluidic channel bottom of the fluidic channel. In some embodiments, the substrate comprises silicon, fused silica, glass, a polymer, a metal, an elastomer, polydimethylsiloxane, agarose, a hydrogel, or a combination thereof. In some embodiments, the cartridge is an inseparable assembly in which the flow cell is irreversibly attached to the housing. In some cases, the flow cell is fabricated by bonding a microwell substrate to a fluidic substrate.

[0066] Flow cells can be fabricated using a variety of materials known to those skilled in the art. Generally, the choice of material used will depend on the choice of fabrication technique used, and vice versa. Examples of suitable materials include, but are not limited to, silicon, fused silica, glass, any of a variety of polymers, such as polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, metals (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), non-stick materials such as Teflon (PTFE), or combinations of these materials. Cyclic olefin polymers (COP) can include Zeonor 1020R or Zeonor 1060R.

[0067] Microwell Array In some embodiments, a microwell can comprise a small reaction chamber of a defined volume. In some embodiments, a microwell can capture one or more cells. In some embodiments, a microwell can capture only one cell. In some embodiments, a microwell can capture one or more solid supports (e.g., beads). In some embodiments, a microwell can capture only one solid support. In some embodiments, a microwell captures a single cell and a single solid support (e.g., bead). In some embodiments, a microwell can accommodate a single particle (e.g., a cell or a bead). In some embodiments, a microwell can accommodate two different particles (e.g., a cell and a bead).

[0068] Microwells can be fabricated in a variety of shapes. Non-limiting exemplary well geometries include a cylinder, a cone, a hemisphere, a rectangle, or a polyhedron (e.g., a three-dimensional geometric shape consisting of several planes, e.g., a hexagonal prism, an octagonal prism, an inverted triangular pyramid, an inverted square pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted truncated pyramid). A microwell can have a shape that combines two or more of these geometric shapes. For example, a microwell can be partially cylindrical, with the remainder having the shape of an inverted cone. A microwell can include two side-by-side cylinders, one with a diameter (e.g., approximately corresponding to the diameter of a bead) larger than the other (e.g., approximately corresponding to the diameter of a cell), connected by a vertical channel (i.e., parallel to the cylinder axis) that extends the entire length (depth) of the cylinders. The location of the opening of the microwell can vary. For example, the opening of the microwell can be on the top surface of the substrate. For example, the opening of the microwell can be on the bottom surface of the substrate. The shape of the closed end, e.g., the bottom, of the microwell can vary. For example, the closed end of the microwell can be flat. For example, the closed end of the microwell can be curved (e.g., convex or concave). The shape and / or size of the microwell can be determined based on the type of cell or solid support to be captured within the microwell. In some embodiments, the microwell can have a non-circular cross-section (e.g., square or hexagonal) in the plane of the substrate.

[0069] Microwells can be manufactured in a variety of sizes. Microwell size can be characterized, for example, in terms of the diameter and / or depth of the microwell. The diameter of a microwell can refer to the largest circle that can be inscribed within a planar cross-section of the microwell shape. The diameter of a microwell, in some embodiments, can range from about 1 to about 10 times the diameter of a cell or solid support to be captured within the microwell. In some embodiments, the diameter of a microwell can be 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times the diameter of a cell or solid support to be captured within the microwell, or a multiple of any number or range between any two of these values, or can be approximately a multiple thereof. In some embodiments, the diameter of a microwell can be at least or at most 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times the diameter of a cell or solid support to be captured within the microwell. In some embodiments, the diameter of the microwell may be about 2.5 times the diameter of the cell or solid support to be trapped within the microwell.

[0070] The diameter of a microwell can be specified in terms of absolute dimensions. The diameter of a microwell can range from about 1 nanometer to about 1000 micrometers. In some embodiments, the diameter of a microwell can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or range of diameters between any two of these values, or about these values. In some embodiments, the diameter of the microwells may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers. In some embodiments, the diameter of the microwells may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or range of diameters between any two of these values, or may be about these values. In some embodiments, the diameter of the microwells may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 micrometers, In some embodiments, the diameter of the microwells may be about 30 micrometers.

[0071] The depth of the microwells may be varied, for example, to provide for efficient capture of droplets, such as cells or solid supports, or to provide for efficient exchange of assay buffers and other reagents contained within the wells. The ratio of diameter to depth (i.e., aspect ratio) may be varied so that cells and / or solid supports are not displaced by fluid movement above the microwells as they settle into the microwells. In some embodiments, the depth of the microwells may be less than the diameter of the beads. For example, the depth of the microwells may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%, 100% of the diameter of the beads, or a number or range between any two of these values, or may be about these values. For example, the depth of the microwell can be at least or at most 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%, 100% of the diameter of the bead. In some embodiments, synthetic particles such as beads can protrude outside the microwell.

[0072] In some embodiments, the dimensions of the microwell allow the microwell to accommodate at most one bead. The ratio of the width of the microwell to the diameter of the bead may vary from 1 to 1.9. In some embodiments, the ratio of the width of the microwell to the diameter of the bead may be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or a number or range between or about any two of these values. In some embodiments, the ratio of the width of the microwell to the diameter of the bead may be at least or at most 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9.

[0073] The dimensions of the microwells may vary so that the microwells have sufficient space to accommodate solid supports and cells of various sizes without being displaced by fluid movement above the microwells. The depth of the microwells may range from about 1 to about 10 times the diameter of the cell or solid support to be captured within the microwell. In some embodiments, the depth of the microwells may be 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times the diameter of the cell or solid support to be captured within the microwell, or a multiple of any number or range between any two of these values, or may be about a multiple of these values. In some embodiments, the depth of the microwells may be about 2.5 times the diameter of the cell or solid support to be captured within the microwell.

[0074] The aspect ratio of the width of the microwell to the depth of the microwell may vary, for example, in the range of 0.1 to 2. In some embodiments, the aspect ratio of the width of the microwell to the depth of the microwell may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or a number or range between any two of these values, or may be about these values. In some embodiments, the aspect ratio of the width of the microwell to the depth of the microwell may be at least or at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.

[0075] The depth of a microwell can be specified in terms of its absolute dimension. For example, the depth of a microwell can range from about 1 nanometer to about 1000 micrometers. In some embodiments, the depth of a microwell can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or range between any two of these values, or about these values. In some embodiments, the depth of the microwells may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 micrometers, or a number or range between or about any two of these values. In some embodiments, the depth of the microwells can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 micrometers. In some embodiments, the depth of the microwells ranges from 20 micrometers to 60 micrometers, such as from 25 micrometers to 55 micrometers, such as from 30 micrometers to 50 micrometers, and including from 45 micrometers to 49 micrometers. In some cases, the depth of the microwells is 48 micrometers.

[0076] The volume of a microwell may vary, for example, from about 1 picoliter to about 1,000 microliters, hi some embodiments, the volume of a microwell may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 picoliters, or a number or range of volumes between any two of these values, or may be about these values. In some embodiments, the volume of a microwell may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 picoliters. In some embodiments, the volume of a microwell may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 nanoliters, or a number or range of volumes between or about any two of these values. In some embodiments, the volume of a microwell may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nanoliters. In some embodiments, the volume of a microwell may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 microliters, or a number or range of volumes between or about any two of these values. In some embodiments, the volume of a microwell can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.In some embodiments, the volume of the microwell may be about 1 microliter.

[0077] The volume of a microwell can be characterized in terms of variation in volume from one microwell to another. The coefficient of variation (expressed as a percentage) of the volume of a microwell can range from about 1% to about 100%. The coefficient of variation of the volume of a microwell can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, or can be about these values. The coefficient of variation of the volume of a microwell can be at least or at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the coefficient of variation of the microwell volume may be about 2.5%.

[0078] The ratio of the volume of the microwell to the surface area of ​​the bead (or the surface area of ​​the solid support to which the stochastic barcode oligonucleotides can be attached) can vary, for example, from about 2.5 to about 1,520 micrometers. In some embodiments, the ratio can be 2.5, 5, 10, 100, 500, 750, 1,000, 1,520 micrometers, or a number or range between or about any two of these values. In some embodiments, the ratio can be at least or at most 2.5, 5, 10, 100, 500, 750, 1,000, or 1,520 micrometers. In some embodiments, the ratio can be about 67.5 micrometers.

[0079] The microwells can be arranged in one-, two-, or three-dimensional arrays. A three-dimensional array can be achieved, for example, by stacking a series of two or more two-dimensional arrays, e.g., by stacking two or more substrates comprising microwell arrays.

[0080] The pattern and spacing between the microwells may be varied to optimize the efficiency of capturing a single cell and a single solid support (e.g., a bead) in each well, as well as to maximize the number of wells per unit area of ​​the array. The microwells can be distributed according to a variety of random or non-random patterns. For example, they can be distributed completely randomly across the surface of the array substrate, or can be arranged in a square grid, rectangular grid, hexagonal grid, etc.

[0081] The center-to-center distance or spacing between wells can vary from about 1 micrometer to about 1,000 micrometers, in some embodiments, the center-to-center distance between wells can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 micrometers, or a number or range of distances between or about any two of these values. In some embodiments, the center-to-center spacing between wells can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 micrometers, hi some embodiments, the center-to-center spacing between wells can be about 4,890 micrometers.

[0082] The distance or spacing between the edges of the microwells can vary from about 1 micrometer to about 1,000 micrometers, in some embodiments, the distance between the edges of the wells can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or range of distances between or about any two of these values. In some embodiments, the distance between the edges of the wells can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 micrometers, hi some embodiments, the distance between the edges of the wells can be about 80 micrometers.

[0083] The microwell array may have, for example, 100 microwells per inch. 2 ~1,000,000 microwells / inch 2 In some embodiments, the density of the microwell array can be in the range of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90 000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000 microwells / inch 2In some embodiments, the density of the microwell array can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 microwells / cm 2 In some embodiments, the density of the microwell array can be at least or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 microwells / cm. 2 In some cases, each microwell array may have 20,000 microwells / cm 2 ~50,000 microwells / cm 2 etc., and 36,000 microwells / cm 2 ~42,000 microwells / cm 2 Contains 10,000 microwells / cm 2 ~60,000 microwells / cm 2 Includes densities in the range of

[0084] The total number of microwells on the substrate can vary based on the well pattern and spacing, as well as the overall dimensions of the array. The number of microwells in the array can vary, for example, from about 96 to about 1,000,000. In some embodiments, the number of microwells in a microarray can be 96, 384, 1,536, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1,000, 00, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 10 8 , 10 9 In some embodiments, the number of microwells in a microarray may be at least or at most 96, 384, 1536, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 9000, 0, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 10 8 , 10 9In some embodiments, the number of microwells in a microwell array may be about 96. In some embodiments, the number of microwells may be about 150,000. In some cases, each microwell array comprises between 100,000 microwells and 500,000 microwells, such as between 200,000 microwells and 250,000 microwells, and including between 250,000 microwells and 300,000 microwells. In some cases, each microwell array comprises at least 220,000 microwells per lane.

[0085] The microwells of the microwell array may further have any suitable pitch, where pitch represents the amount of separation between microwells. For example, in some cases, the calculation of pitch includes the well cross-sectional area and wall separation. In some cases, the pitch is in the range of 1 μm to 500 μm, such as 5 μm to 450 μm, such as 10 μm to 400 μm, such as 15 μm to 350 μm, such as 20 μm to 300 μm, such as 25 μm to 250 μm, such as 30 μm to 200 μm, such as 35 μm to 150 μm, and 40 μm to 100 μm. In some cases, the microwells have a pitch of 20 μm or greater, such as 25 μm or greater, such as 30 μm or greater, such as 35 μm or greater, such as 35 μm or greater, such as 40 μm or greater, such as 45 μm or greater, such as 49 μm or greater, such as 50 μm or greater, and including 55 μm or greater. In one example, the microwell array has a well pitch of 49 μm (39 μm well cross section + 10 μm wall separation).

[0086] Microwell arrays can include surface features between the microwells designed to aid in guiding the cells and solid supports into the wells and / or to prevent their settling onto the inter-well surface. Non-limiting examples of suitable surface features include, but are not limited to, dome-shaped, ridged, or peak-shaped surface features that surround the wells or span the inter-well surface.

[0087] The microwells can be fabricated using any of several fabrication techniques, non-limiting examples of which include bulk micromachining techniques such as photolithography and wet chemical etching, plasma etching, or deep reactive ion etching, micromolding and microembossing, laser micromachining, 3D printing or other direct-write manufacturing processes using curable materials, and similar techniques.

[0088] Microwell arrays can be fabricated from a variety of substrate materials. The choice of material may depend on the choice of fabrication technique, or vice versa. Non-limiting examples of suitable materials include fused silica, glass, polymers (e.g., agarose, gelatin, hydrogels, polydimethylsiloxane (PDMS) elastomers, polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimides, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resins, thiol-ene based resins, metals or metal films (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), and the like. Hydrophilic materials (e.g., enhance wettability and promote cell and It may be desirable for the fabrication of microwell arrays (to minimize nonspecific binding of other biological materials). Hydrophobic materials that can be treated or coated (e.g., by oxygen plasma treatment or grafting of a polyethylene oxide surface layer) can be used for the fabrication of microwell arrays. The use of porous hydrophilic materials for the fabrication of microwell arrays may be desirable to facilitate capillary wicking / venting of gases or bubbles trapped within the device. Microwell arrays can be fabricated from a single material. Microwell arrays can include two or more different materials bonded or mechanically joined to each other.

[0089] Substrates can have a variety of shapes and sizes. For example, the shape (or footprint) of the substrate in which the microwells are fabricated can be square, rectangular, circular, or irregular. The size of a substrate can be characterized by its width, length, and depth.

[0090] The thickness of the substrate on which the microwells are fabricated can range from about 0.1 mm thick to about 10 mm thick, or more. The thickness of the microwell array substrate can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm, or any number or range between or about any two of these values. The thickness of the microwell array substrate can be at least or at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 mm. The thickness of the microwell array substrate can be about 1 mm thick. The thickness of the microwell array substrate can be any value within these ranges; for example, the thickness of the microwell array substrate can be about 0.2 mm to about 9.5 mm.

[0091] Various surface treatments and surface modification techniques can be used to modify the properties of the microwell array surface. Examples may include, but are not limited to, oxygen plasma treatment to make hydrophobic material surfaces more hydrophilic, the use of wet or dry etching techniques to smooth or roughen glass and silicon surfaces, the adsorption or grafting of polyethylene oxide or other polymer layers, such as Pluronic®, or bovine serum albumin, to substrate surfaces to make them more hydrophilic and less susceptible to nonspecific adsorption of biomolecules and cells, and the use of silane reactions to graft chemically reactive functional groups to otherwise inert silicon and glass surfaces. Photodeprotection techniques can be used to selectively activate chemically reactive functional groups at specific locations within the array structure; for example, selective addition or activation of chemically reactive functional groups, such as primary amine or carboxyl groups, on the interior walls of the microwells can be used to covalently attach oligonucleotide probes, peptides, proteins, or other biomolecules to the walls of the microwells. The choice of surface treatment or surface modification utilized may depend on the type of surface properties desired and / or the type of material from which the microwell array is fabricated.

[0092] Figures 9A-9C show different depictions of microwells of a flow cell in a cartridge of the present invention. Figure 9A shows a microwell 901 in a flow cell of a multi-microwell array flow cell cartridge according to certain embodiments. As shown in Figure 9A, the microwell array includes hexagonal microwells. Figure 9B provides a detailed depiction of the hexagonal microwells shown in Figure 9A, and Figure 9C shows pitch measurements according to one embodiment of the present invention. As shown in Figure 9C, the wells have a well pitch of 49 μm (39 μm well cross-section + 10 μm wall separation).

[0093] FIG. 10 illustrates fluid flow through a multi-microwell array flow cell cartridge according to certain embodiments. Liquid is introduced into cartridge 1000 via pipette 1001 using inlet 1002. The liquid follows fluid path 1004 and passes through elongated channels 1003 of the flow cell. The flow cell comprises a microwell array such as that described with respect to FIGS. 9A-9C. Single cells can be trapped within the wells. Fluid path 1004 leads to outlet 1005, which is shown as a stepped outlet including a raised portion that the fluid must traverse before being expelled. This prevents siphoning of the liquid.

[0094] cartridge The cartridge may further comprise components designed to create physical or chemical barriers to prevent diffusion (or increase path length and diffusion time) of large molecules to minimize cross-contamination between microwells. Examples of such barriers may include, but are not limited to, a serpentine channel pattern used to deliver cells and solid supports (e.g., beads) to the microwell array, a retractable platen or deformable membrane that is pressed into contact with the surface of the microwell array substrate during a lysis or incubation step, the use of large beads, such as the aforementioned Sephadex beads, to block the openings of the microwells, or the release of an immiscible hydrophobic fluid from a reservoir within the cartridge during a lysis or incubation step to effectively separate and compartmentalize each microwell within the array.

[0095] The cartridge can be manufactured using a variety of techniques and materials known to those skilled in the art. Typically, the cartridge is manufactured as a series of separate component parts that are then assembled using any of several mechanical assembly or joining techniques. Examples of suitable manufacturing techniques include, but are not limited to, conventional machining, CNC machining, injection molding, thermoforming, and 3D printing. Once the cartridge components are manufactured, they can be mechanically assembled using screws, clips, etc., or (depending on the selection of materials used) permanently joined using any of a variety of techniques, such as thermal bonding / welding, or any of a variety of adhesives or adhesive films, including epoxy-, acrylic-, silicone-, UV-curable, polyurethane-, or cyanoacrylate-based adhesives.

[0096] Cartridge components can be manufactured using any of a number of suitable materials, including, but not limited to, silicon, fused silica, glass, any of a variety of polymers, such as polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as Teflon (PTFE), metals (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof.

[0097] The cartridge may include an integrated miniature pump or other fluid actuation mechanism for controlling fluid flow through the device. Examples of suitable miniature pumps or fluid actuation mechanisms may include, but are not limited to, electromechanical or pneumatically actuated miniature syringe or plunger mechanisms, membrane diaphragm pumps actuated pneumatically or by an external piston, pneumatically actuated reagent pouches or bladders, or electroosmotic pumps.

[0098] The cartridge may contain miniature valves for compartmentalizing pre-filled reagents or for controlling fluid flow through the device. Examples of suitable miniature valves may include, but are not limited to, one-shot "valves" fabricated using wax or polymer plugs that can be melted or dissolved, or polymer membranes that can be punctured, pinch valves constructed using a deformable membrane and pneumatic, magnetic, electromagnetic, or electromechanical (solenoid) actuation, one-way valves constructed using a deformable membrane flap, and miniature gate valves.

[0099] The cartridge may include a vent to provide an escape path for trapped air or gas, such as CO or N. The vent may be constructed according to a variety of techniques, for example, using a porous plug of polydimethylsiloxane (PDMS) or other hydrophobic material that allows capillary wicking of air or gas but blocks penetration by water.

[0100] The mechanical interface features of the cartridge can provide highly accurate and reproducible positioning while allowing for easy removal of the cartridge from the instrument system. Suitable mechanical interface features can include, but are not limited to, alignment pins, alignment guides, mechanical stops, etc. Mechanical design features can include relief features for locating external devices, such as magnets or optical components, in close proximity to the microwell array chamber.

[0101] The cartridge may include temperature control components or thermal interface features for coupling to an external temperature control module. Examples of suitable temperature control elements may include, but are not limited to, resistive heating elements, miniature infrared emitting light sources, Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, etc. The thermal interface features may be fabricated from materials that are good thermal conductors (e.g., copper, gold, silver, etc.) and may include one or more flat surfaces that may make good thermal contact with an external heating or cooling block.

[0102] The cartridge can include optical interface features for use in optical imaging or spectroscopic interrogation of the microwell array. The cartridge can include an optically transparent window, such as in the microwell substrate itself or on the side of the flow cell or microarray chamber opposite the microwell array, fabricated from a material that meets the spectral requirements of the imaging or spectroscopic technique used to probe the microwell array. Examples of suitable optical window materials can include, but are not limited to, glass, fused silica, polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic olefin polymer (COP), or cyclic olefin copolymer (COC).

[0103] Cartridge Carrier Embodiments of the present invention may also include a carrier for the cartridge. The carrier may be used, for example, to transport the cartridge. In some cases, the cartridge carrier is configured to support imaging of cartridge / flow cell contents in any of the microwell lanes during an experiment. In certain cases, the cartridge includes one or more kinematic mounts configured to orient the cartridge carrier when placed within the cartridge carrier within an analyzer (e.g., a BD Rhapsody™ scanner). In some such examples, the number of kinematic mounts within the cartridge carrier may range from 1 to 5, including 2 to 4. In some cases, the cartridge carrier has three kinematic mounts. The cartridge carrier may include one or more relief portions. The relief portions may be used to install or remove the cartridge from the holder. In some cases, the cartridge carrier includes chamfered corners to allow the cartridge to be placed within the carrier in a single orientation. In certain versions, the cartridge carrier includes a drip receptacle configured to isolate droplets exiting the cartridge outlet.

[0104] The cartridge carrier may be configured for use with a single-lane cartridge (such as those described in U.S. Pat. Nos. 10,634,691 and 11,061,043) or a multi-microwell-array-flow cell cartridge of the present invention. In some cases, the cartridge carrier may be configured for use with a single-lane cartridge. In such cases, the cartridge carrier may include a drip receptacle positioned to isolate droplets exiting a single outlet of the single-lane cartridge. In other cases, the cartridge carrier may be configured for use with a multi-microwell-array-flow cell cartridge of the present invention. In such cases, the cartridge carrier may include a drip receptacle positioned to isolate droplets exiting each outlet of the multi-microwell-array-flow cell cartridge of the present invention. In still other cases, the cartridge carrier is configured for use with both a single-lane cartridge and a multi-microwell-array-flow cell cartridge of the present invention. In some such cases, the cartridge carrier includes both a drip receptacle positioned to isolate droplets exiting each outlet of a multi-microwell-array-flow cell cartridge of the present invention, and a drip receptacle positioned to isolate droplets exiting a single outlet of a single-lane cartridge.

[0105] The cartridge carrier may be constructed from any convenient material. In some cases, the cartridge carrier is constructed from metallic materials, including, but not limited to, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In selected versions, the cartridge carrier is constructed from hard-anodized 6061-T6 aluminum impregnated with Class III PTFE.

[0106] A cartridge carrier of the present invention is shown in Figures 11A-11H. Figures 11A-11B show different side views of cartridge carrier 1100. Figure 11C shows a top view of cartridge carrier 1100. As shown in Figure 11C, cartridge 1100 includes a relief portion 1101 configured for installation or removal of the cartridge from a holder. Cartridge 1100 also includes drip receptacles 1102a-b. Drip receptacle 1102a is positioned to isolate droplets exiting a single outlet of a single-lane cartridge, while drip receptacle 1102b is positioned to isolate droplets exiting each outlet of a multi-microwell-array-flow cell cartridge of the present invention. Cartridge bias relief 1103 and chamfered corners 1104 are also shown. Figure 11D shows a bottom view of cartridge carrier 1100, revealing kinematic mount 1105. 11E-11F show cartridge carrier 1100 viewed at a 45° angle, while FIGS. 11G-11H show side views of cartridge carrier 1100. FIG.

[0107] Sample Collection Container Holder As noted above, aspects of the present invention also include sample container holders. The subject sample container holders are configured to receive multiple sample collection containers for collecting analytes from a multi-microwell array flow cell cartridge (e.g., as described above). The sample collection container holder may be configured to accommodate any suitable number of sample collection containers, including, for example, from 2 to 10 and from 7 to 9. In some cases, the system includes eight sample collection containers. In selected embodiments, the sample collection container holder includes a counterweight configured to maintain the sample collection container in an upright position. In certain embodiments, the sample collection container holder has a shape complementary to the cellular analysis system so that the sample collection container holder can be received by the cellular analysis system in a single orientation. In other words, the sample collection container holder may have a poka-yoke design. In some cases, the sample collection container holder has rounded edges.

[0108] The sample vessel holder may be constructed from any of several suitable materials, including, but not limited to, silicon, fused silica, glass, any of a variety of polymers, such as polydimethylsiloxane (PDMS; an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as Teflon (PTFE), metals (e.g., aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof.

[0109] 12A-12C illustrate a sample collection container holder of the present invention according to certain embodiments. As shown in FIG. 12A, sample collection container holder 1201 is configured to hold eight sample collection containers 1202a-1202h, shown as test tubes. FIG. 12B illustrates a top view of sample collection container holder 1201. As indicated by the rounded edges, sample collection container holder 1201 is shaped so that it can only be inserted into the drawer of a system of the present invention in a single orientation. As shown in FIG. 12C, sample collection container holder 1201 includes a counterweight 1203 configured to maintain the sample collection holder in an upright position.

[0110] Method for processing a cell sample As described above, aspects of the present invention include methods for processing a cellular sample. The subject methods include introducing a multi-microwell array flow cell cartridge into a system including a tray configured to receive the multi-microwell array flow cell cartridge and a collection magnet assembly configured to apply a uniform magnetic force to a flow cell of the multi-microwell array flow cell cartridge when in an active position. The method also includes loading a sample into the multi-microwell array flow cell cartridge, actuating the collection magnet assembly to an active position to apply a uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to create a processed sample, and collecting the processed sample from the multi-microwell array flow cell cartridge.

[0111] In certain cases, the method includes loading multiple different samples into different flow cells of the cartridge. The number of samples can vary, and in some cases can range from 2 to 10 (e.g., 8). Methods according to certain embodiments include loading the multi-well cartridge with a sample liquid followed by loading the multi-well cartridge with a lysis buffer. In some versions, the method includes loading the multi-well cartridge with a lysis buffer without using a lysis magnet. Methods according to selected versions of the invention include loading barcoded beads into the multi-well cartridge prior to actuation of the collection magnet assembly. In certain cases, the barcoded beads can include a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain. In selected embodiments, the target capture domain is a poly(T) sequence. In certain instances, the nucleic acid barcode further includes a unique molecular index (UMI).

[0112] In certain instances, the method includes applying a uniform magnetic force from a position above the tray. As described above, a system that can be used in the method includes a tray configured to receive a multi-microwell array flow cell cartridge and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cells of the cartridge when in an active position. In some instances, the retrieval magnet assembly is configured to apply a uniform magnetic force from a position above the tray. In certain instances, the uniform magnetic force may be a magnetic field in the range of 650 Gauss to 1325 Gauss. In certain examples, the retrieval magnet assembly is configured to apply a uniform magnetic force via multiple magnets. In some such instances, the multiple magnets have alternating polarities. The number of magnets in the retrieval magnet assembly may vary, in some instances ranging from 1 to 10 (e.g., 4). The type of magnet in the retrieval magnet assembly may also vary. In some instances, the magnets are rare earth magnets (e.g., neodymium magnets and / or samarium-cobalt magnets). The shape of the magnets may also vary. In some instances, the multiple magnets are bar magnets. In other cases, the multiple magnets are ring magnets. In some such cases, the multiple magnets are arranged in a bull's-eye configuration. The systems of the present invention may be configured to adjust the arrangement of the retrieval magnet assembly to process the cell sample. In some embodiments, the retrieval magnet assembly is operable between an operative position, in which the retrieval magnet assembly is positioned adjacent to the tray, and a non-operative position, in which the retrieval magnet is positioned further away from the tray relative to the operative position.

[0113] In some versions, the method includes using a sample collection container holder configured to receive multiple sample collection containers for collecting analytes from the cartridge. In some such versions, the sample collection container holder includes a counterweight configured to maintain the sample collection container in an upright position. The system and the sample collection container holder may also have complementary shapes so that the sample collection container holder can be received in the system in a single orientation. In some aspects, the system also includes multiple sample collection containers. The number of sample collection containers may vary and, in some cases, may range from 2 to 10 (e.g., 8). In particular examples, the system includes a number of sample collection containers that matches the number of flow cells in the cartridge. In some embodiments, the system includes a waste collection container for collecting liquid waste from the multi-microwell-array-flow cell cartridge. In some cases, the method includes an interlock configured to prevent collection of sample liquid into the waste collection container when the collection magnet assembly is in the activated position. In some cases, the tray includes a latch for holding the multi-microwell-array-flow cell cartridge. In selected versions, the system does not include a lysis magnet located below the tray.

[0114] As described above, the methods of the present invention involve the use of a multi-microwell array flow cell cartridge. A multi-microwell array flow cell cartridge for use in the methods includes multiple fluid lanes, each of which includes an inlet for receiving liquid, a flow cell comprising a microwell array, and an outlet for discharging liquid. In certain cases, the multi-microwell array flow cell cartridge includes a number of fluid lanes ranging from 2 to 10 (e.g., 8). In some versions, each outlet is stepped to prevent siphoning of liquid from the flow cell. In some embodiments, each flow cell includes an elongated channel. The length of the elongated channel may vary, in some cases ranging from 50 mm to 100 mm. The number of microwells within each microwell array may also vary, in some cases ranging from 250,000 microwells to 300,000 microwells. The density of microwells within each microwell array may vary, in some cases ranging from 36,000 microwells / cm. 2 ~42,000 microwells / cm 2 may be in the range of

[0115] The type of analysis performed in the method may vary as needed. In some cases, the method includes generating a sequence-ready nucleic acid library from the processed sample. In certain embodiments, the sequence-ready nucleic acid library is sequenceable by using a next-generation sequencing protocol. In further embodiments, the method is a method of genomic, epigenomic, transcriptomic, or proteomic analysis. In selected versions, the method is a method of multi-omic analysis, such as when the multi-omic analysis includes at least transcriptomic and proteomic analysis.

[0116] Probabilistic Barcodes In some embodiments, the method includes barcoding (e.g., stochastic barcoding). Stochastic barcoding is described, for example, in U.S. Patent Application Publication No. 20150299784, International Publication No. 2015031691, and Fu et al., Proc Natl Acad Sci USA, 108(22):9026-31, May 31, 2011, the contents of which are incorporated herein in their entireties. Briefly, a stochastic barcode can be a polynucleotide sequence that can be used to stochastically label (e.g., barcode, tag) a target. A stochastic barcode can include one or more labels. Exemplary labels can include a universal label, a cellular label, a molecular label, a sample label, a plate label, a spatial label, and / or a pre-spatial label. A stochastic barcode can include a 5' amine that can link the stochastic barcode to a solid support. A stochastic barcode can include a universal label, a dimensional label, a spatial label, a cellular label, and / or a molecular label. The order of the various labels (including, but not limited to, the universal label, dimensional label, spatial label, cellular label, and molecular label) within the stochastic barcode may vary. For example, the universal label may be the 5'-most label and the molecular label may be the 3'-most label. The spatial label, dimensional label, and cellular label may be in any order. In some embodiments, the universal label, spatial label, dimensional label, cellular label, and molecular label are in any order.

[0117] The stochastic barcodes can be from a "non-depleting reservoir," which is a pool of stochastic barcodes composed of many different labels. The non-depleting reservoir can contain a large number of different stochastic barcodes, so that when the non-depleting reservoir is associated with a pool of targets, each target is likely to be associated with a unique stochastic barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the copy number of the same target molecule in the collection compared to the diversity of the labels. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected stochastic barcodes then allows for calculation of the number of target molecules present in the original collection or sample. When the ratio of the copy number of target molecules present to the number of unique stochastic barcodes is low, the labeled target molecules are highly unique (i.e., the likelihood of more than one target molecule being labeled with a given label is extremely low).

[0118] Labels, e.g., cellular labels, can include a unique set of nucleic acid subsequences of a defined length, e.g., seven nucleotides each (equivalent to the number of bits used in some Hamming error-correcting codes), which can be designed to provide error correction capabilities. A set of error-correcting subsequences comprising seven nucleotide sequences can be designed so that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases); for example, a set of error-correcting subsequences can be designed to exhibit a genetic distance of three nucleotides. In this case, review of the error-correcting sequences in a set of sequence data for a labeled target nucleic acid molecule (described more fully below) allows amplification or sequencing errors to be detected or corrected. In some embodiments, the lengths of the nucleic acid subsequences used to create the error-correcting codes can vary; for example, they can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, or 50 nucleotides in length, or a number or range of nucleotides between or about any two of these values. In some embodiments, nucleic acid subsequences of other lengths can be used to create error correcting codes.

[0119] The stochastic barcode can include a target-binding region. The target-binding region can interact with a target in a sample. The target can be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, short interfering RNA (siRNA), RNA degradation products, RNAs each containing a poly(A) tail, and any combination thereof. In some embodiments, the multiple targets can include deoxyribonucleic acid (DNA).

[0120] In some embodiments, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more labels of the stochastic barcode (e.g., universal label, dimensional label, spatial label, cellular label, and molecular label) can be separated from another one or two of the remaining labels of the stochastic barcode by a spacer. The spacer can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides. In some embodiments, none of the labels of the stochastic barcode are separated by a spacer.

[0121] The stochastic barcode can include one or more universal labels, one or more dimensional labels, one or more spatial labels, one or more cellular labels, one or more molecular labels, one or more target binding regions, or any combination thereof.

[0122] The one or more universal labels can be the same for all stochastic barcodes in a set of stochastic barcodes attached to a given solid support (e.g., a bead), or can be the same for all stochastic barcodes attached to multiple beads. A universal label can include a nucleic acid sequence that can hybridize to a sequencing primer. A universal label can include a nucleic acid sequence that can hybridize to a PCR primer, or a nucleic acid sequence that can hybridize to a sequencing primer and a PCR primer. A nucleic acid sequence of a universal label that can hybridize to a sequencing or PCR primer can be referred to as a primer binding site. A universal label can include a sequence that can be used to initiate transcription of a stochastic barcode. A universal label can include a sequence that can be used to extend a stochastic barcode or a region within a stochastic barcode.

[0123] Dimensional labels can include nucleic acid sequences that provide information about the dimension in which stochastic labeling occurred. For example, dimensional labels can provide information about the time at which a target was stochastically barcoded. Dimensional labels can be associated with the time of stochastic barcoding in a sample. Dimensional labels can be activated at the time of stochastic labeling. Different dimensional labels can be activated at different times. Dimensional labels provide information about the order in which targets, groups of targets, and / or samples were stochastically barcoded. For example, a population of cells can be stochastically barcoded in the G0 phase of the cell cycle. Cells can be pulsed again with stochastic barcodes in the G1 phase of the cell cycle. Cells can be pulsed again with stochastic barcodes in the S phase of the cell cycle, etc. The stochastic barcodes in each pulse (e.g., each phase of the cell cycle) can include different dimensional labels. In this way, dimensional labels provide information about which targets were labeled in which phase of the cell cycle. Dimensional labels can interrogate many different biological time periods. Exemplary biological times may include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a population of cells) may be stochastically labeled before and / or after treatment with a drug and / or therapy. Changes in the copy number of distinct targets may indicate the sample's response to the drug and / or therapy.

[0124] solid support In some embodiments, the stochastic barcodes disclosed herein can be associated with a solid support. The solid support can be, for example, a synthetic particle. In some embodiments, some or all of the molecular labels (e.g., first molecular labels) of a plurality of stochastic barcodes (e.g., a first plurality of stochastic barcodes) on a solid support differ by at least one nucleotide. The cellular labels of stochastic barcodes on the same solid support can be the same. The cellular labels of stochastic barcodes on different solid supports can differ by at least one nucleotide. For example, a first cellular label of a first plurality of stochastic barcodes on a first solid support can have the same sequence, and a second cellular label of a second plurality of stochastic barcodes on a second solid support can have the same sequence. The first cellular label of a first plurality of stochastic barcodes on a first solid support and the second cellular label of a second plurality of stochastic barcodes on a second solid support can differ by at least one nucleotide. The cellular labels can be, for example, about 5 to 20 nucleotides in length. Molecular labels can be, for example, about 5 to 20 nucleotides in length.

[0125] The synthetic particles can be, for example, beads. The beads can be, for example, silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic materials, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.

[0126] For example, cells, such as single cells, can be introduced into multiple microwells of a microwell array, followed by the introduction of beads into multiple microwells of the microwell array. Each microwell can contain one bead. The beads can contain multiple stochastic barcodes. The stochastic barcodes can include 5' amine regions attached to the beads. The stochastic barcodes can include universal labels, molecular labels, target binding regions, or any combination thereof.

[0127] The stochastic barcodes disclosed herein can be associated (e.g., attached) to a solid support (e.g., a bead). Each stochastic barcode associated with a solid support can comprise a molecular label selected from a group comprising at least 100 or 1000 molecular labels having a unique sequence. In some embodiments, different stochastic barcodes associated with a solid support can comprise molecular labels of different sequences. In some embodiments, a percentage of the stochastic barcodes associated with a solid support comprise the same cellular label. For example, the percentage can be about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or can be about these values. As another example, the percentage can be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, the stochastic barcodes associated with a solid support can have the same cellular label. The stochastic barcodes associated with different solid supports can have different cellular labels selected from a group including at least 100 or 1000 cellular labels having unique sequences.

[0128] In some cases, the barcoded beads comprise a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain. In some cases, the target capture domain is a poly(T) sequence. In certain versions, the nucleic acid barcode further comprises a unique molecular index (UMI). Stochastic barcodes with unique molecular labels (also referred to as molecular indexes (MIs)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA), can correct biases induced by PCR and library preparation steps by using molecular labels (MLs) to label mRNA during reverse transcription (RT). In some embodiments, the method includes using a non-depleting pool of stochastic barcodes with a large number of unique molecular labels (e.g., 6561 to 65536) on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. The stochastic barcode can include a universal PCR priming site. During RT, target gene molecules react randomly with the stochastic barcode. Each target molecule can hybridize to the stochastic barcode to generate a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from the microwells of the microwell plate can be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to calculate the number of reads, the number of stochastic barcodes with unique molecular labels, and the number of mRNA molecules.

[0129] In some embodiments, stochastic barcoding of multiple targets in a sample can be performed using a solid support comprising multiple synthetic particles associated with multiple stochastic barcodes. In some embodiments, the solid support can include multiple synthetic particles associated with multiple stochastic barcodes. The spatial labeling of the multiple stochastic barcodes on various solid supports can differ by at least one nucleotide. The solid support can include multiple stochastic barcodes, for example, in two or three dimensions. The synthetic particles can be beads. The beads can be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support can include a polymer, matrix, hydrogel, needle array device, antibody, or any combination thereof. In some embodiments, the solid support can be free-floating. In some embodiments, the solid support can be embedded in a semi-solid or solid array. The stochastic barcodes do not have to be associated with a solid support. The stochastic barcodes can be individual nucleotides. The stochastic barcodes can be associated with a substrate.

[0130] As used herein, the terms "tethered," "attached," and "immobilized" are used interchangeably and can refer to covalent or non-covalent means for attaching a stochastic barcode to a solid support. Any of a variety of different solid supports can be used as the solid support for attaching a pre-synthesized stochastic barcode or for in situ solid-phase synthesis of a stochastic barcode.

[0131] In some embodiments, the solid support is a bead. Beads can include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures to which nucleic acids can be immobilized (e.g., covalently or non-covalently). Beads can be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. Beads can be or include discrete particles that are spherical (e.g., microspheres) or have non-spherical or irregular shapes, such as cubes, rectangular prisms, pyramidal, cylindrical, conical, oval, or discoidal shapes. In some embodiments, beads can be non-spherical.

[0132] Beads can comprise a variety of materials, including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (FeO; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogel, polymer, cellulose, nylon, and any combination thereof. In some embodiments, the beads (e.g., beads having stochastic labels attached thereto) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0133] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range of diameters between any two of these values, or about these values.

[0134] The diameter of the bead can be related to the diameter of the well of the substrate. In some embodiments, the diameter of the bead can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% longer or shorter than the diameter of the well, or a number or range between any two of these values, or can be about these values. The diameter of the bead can be related to the diameter of a cell (e.g., a single cell captured by a well of the substrate). In some embodiments, the diameter of the bead can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% longer or shorter than the diameter of the cell, or a number or range between any two of these values, or can be about these values.

[0135] The beads can be attached to and / or embedded in a substrate. The beads can be attached to and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present on the stochastic barcode on the beads, which can serve as a positional address.

[0136] Examples of beads may include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag™ Carboxyl-Terminated Magnetic Beads.

[0137] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one fluorescent optical channel or multiple optical channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be distinguishable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with them. For example, the beads can include a stochastic barcode. The beads can change size, for example, by swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible.

[0138] The solid support (e.g., beads) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging the beads.

[0139] Probabilistic barcoding methods Provided herein are methods for estimating the number of distinct targets at distinct locations in a physical sample (e.g., a tissue, an organ, a tumor, a cell). The methods can include placing a stochastic barcode proximate to the sample, lysing the sample, associating distinct targets with the stochastic barcode, amplifying the targets, and / or digitally counting the targets. The methods can further include analyzing and / or visualizing information obtained from the spatial labels on the stochastic barcode. In some embodiments, the methods include visualizing a plurality of targets in the sample. Mapping the plurality of targets on a map of the sample can include generating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps can be generated before or after stochastically barcoding a plurality of targets in the sample. Visualizing a plurality of targets in the sample can include mapping the plurality of targets on a map of the sample. Mapping the plurality of targets on a map of the sample can include generating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps can be generated before or after stochastically barcoding a plurality of targets in the sample. In some embodiments, the two-dimensional and three-dimensional maps can be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional or three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0140] In some embodiments, stochastically barcoding the plurality of targets comprises hybridizing a plurality of stochastic barcodes to the plurality of targets to create the stochastically barcoded targets. Stochastically barcoding the plurality of targets can comprise generating an indexed library of the stochastically barcoded targets. Generating an indexed library of the stochastically barcoded targets can be performed using a solid support comprising a plurality of stochastic barcodes.

[0141] The present disclosure provides methods for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample comprising a thin section of cells, organ, or tissue can be contacted with a stochastic barcode. For example, cells can be contacted by gravity flow, which allows the cells to settle and form a monolayer. The sample can be a thin section of tissue. The thin section can be placed on a substrate. The sample can be one-dimensional (e.g., forming a flat surface). The sample (e.g., cells) can be spread across the substrate, for example, by growing / culturing the cells on the substrate.

[0142] When the stochastic barcode is in proximity to the target, the target can hybridize to the stochastic barcode. The stochastic barcodes can be contacted in a non-depletable ratio so that each distinct target can be associated with a distinct stochastic barcode of the present disclosure. To ensure efficient association between the target and the stochastic barcode, the target can be cross-linked to the stochastic barcode.

[0143] cell lysis After distribution of cells and stochastic barcodes, the cells can be lysed to liberate target molecules. Cell lysis can be achieved by any of a variety of means, such as chemical or biochemical means, osmotic shock, or thermal, mechanical, or optical lysis. Cells can be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of targets with stochastic barcodes, the diffusion rate of target molecules can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0144] In some embodiments, the sample can be lysed using filter paper, which can be immersed at the top in a lysis buffer, and the filter paper can be applied to the sample with pressure that can facilitate lysis of the sample and hybridization of the sample's targets to the substrate.

[0145] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M Tris HCl or more. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M Tris HCl or more. The lysis buffer can include about 0.1 M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can include a salt (e.g., LiCl). The concentration of the salt in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more. The concentration of the salt in the lysis buffer can be up to about 0.1, 0.5, or 1 M or more. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5 M. The lysis buffer can include a detergent (e.g., SDS, Li dodecyl sulfate, triton X, Tween, NP-40). The concentration of the detergent in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or more. The concentration of detergent in the lysis buffer can be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or more. In some embodiments, the concentration of detergent in the lysis buffer is about 1% Li-dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent used, the shorter the time required for lysis. The lysis buffer can include a chelating agent (e.g., EDTA, EGTA).The concentration of the chelating agent in the lysis buffer can be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of the chelating agent in the lysis buffer can be up to about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer can include a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer can be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer can be up to about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can include about 0.1 M TrisHCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0146] Lysing can be carried out at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysing can be carried out for about 1, 5, 10, 15, or 20 minutes or more. Lysed cells can contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules. Lysed cells can contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules.

[0147] Attaching probabilistic barcodes to target nucleic acid molecules After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules can be randomly associated with the co-localized stochastic barcodes on the solid support. The association can involve hybridization of the target recognition region of the stochastic barcode with a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the stochastic barcode can interact with the poly(A) tail of the target). The assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization can be selected to promote the formation of specific, stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with (e.g., hybridized to) multiple probes on a substrate. If the probes contain oligo(dT), mRNA molecules can hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide can act as a primer for first-strand synthesis of cDNA molecules. For example, mRNA molecules can hybridize to stochastic barcodes on beads. For example, a single-stranded nucleotide fragment can hybridize to a target binding region of a stochastic barcode.

[0148] The attachment can further include ligating the target recognition region of the stochastic barcode with a portion of the target nucleic acid molecule. For example, the target binding region can include a nucleic acid sequence that can specifically hybridize to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure can further include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to create the restriction site overhang. The stochastic barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to join the two fragments.

[0149] For example, labeled targets from multiple cells (or multiple samples) (e.g., target-barcode molecules) can then be pooled, e.g., into a tube. The labeled targets can be pooled, e.g., by collecting the stochastic barcodes and / or beads to which the target-barcode molecules are attached.

[0150] Solid support-based collection and recovery of the attached target-barcode molecules can be implemented through the use of magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling the labeled target nucleic acid molecules from multiple cells.

[0151] 13 shows a non-limiting example of a workflow 1300 that may be performed using the system and multi-microwell-array-flow cell cartridge of the present invention. Workflow 1300 describes steps that are performed after the multi-microwell-array-flow cell cartridge is placed in a tray and a sample collection container is placed in a drawer. Additional steps of inserting the cartridge into the tray and / or inserting a sample collection container or sample collection container holder may be required before workflow 1300 begins if these components are not already in their respective positions.

[0152] Additionally, workflow 1300 describes steps that are performed with the retrieval magnet assembly actuator in its inactive position and the drawer actuator in the waste collection position at the start of workflow 1300. Those skilled in the art will recognize that additional steps may be required to move the actuators to the start of the workflow.

[0153] Workflow 1300 begins at step 1301, where a plurality of cells are introduced into one or more fluid lanes of a cartridge while the cartridge is placed in a system of the invention. In some embodiments, the plurality of cells can be introduced into the cartridge through an inlet (e.g., a gasket). In some embodiments, the plurality of cells can be introduced into the cartridge via a pipette. In some embodiments, the plurality of cells can be placed into a microwell array through a flow cell of the cartridge. In some embodiments, each microwell in the microwell array can capture only a single cell of the plurality of cells. In an alternative embodiment, the plurality of cells can be introduced into the microwell array prior to placing the cartridge in the system.

[0154] After the cells are introduced into the microwell array, in step 1302, a plurality of barcode-containing beads can be introduced into the microwell array. In some embodiments, a plurality of beads can be introduced into the cartridge inlet. In some embodiments, a plurality of beads can be introduced into the cartridge via a pipette. In some embodiments, a plurality of beads can enter the microwell array via a flow cell of the cartridge. In alternative embodiments, a plurality of beads can be introduced into the microwell array before placing the cartridge in a system of the present invention. Those skilled in the art will understand that the order in which the cells are introduced (block 1301) and the beads are introduced (block 1302) is not particularly limited. The two steps can be performed simultaneously or sequentially, and any order is within the scope of the present disclosure. In some embodiments, each microwell in the microwell array can capture only a single bead of the plurality of beads. In some embodiments, each microwell in the microwell array can capture a single cell of the plurality of cells and a single bead of the plurality of beads.

[0155] After the plurality of beads is introduced into the microwell array, cell lysis can be performed in step 1303. In some embodiments, cell lysis can be performed before the plurality of beads is introduced into the microwell array. Cell lysis can be achieved by any of a variety of means described herein. In some embodiments, step 1303 does not include activating a lysis magnet. In some embodiments, beads can be sized such that beads positioned above cells in a microwell can prevent passage of cells from the microwell without removing the beads from the microwell.

[0156] In some embodiments, lysis is performed by introducing a lysis buffer. In some embodiments, a drawer actuator can be used to position the drawer during cell lysis so that a waste collection container is positioned below the cartridge outlet to receive excess buffer flowing through the flow cell. In some embodiments, the introduction of cells and beads results in cells and / or beads located within the flow cell but outside of the microwells. In such embodiments, cells and / or beads located outside of the microwells may be washed into the waste collection container by the lysis buffer.

[0157] In certain embodiments, washing can be performed before, during, or after cell lysis. In some embodiments, a washing fluid can be introduced into the flow cell of the cartridge via an inlet. The washing fluid can flow through the flow cell to remove beads and / or cells that are within the flow cell but outside of the microwells. The removed beads and / or cells can be deposited in a waste collection container aligned with the outlet of the flow cell.

[0158] After cell lysis, the barcode-containing beads can be recovered in step 1304. In some embodiments, the barcode-containing beads are recovered by advancing the recovery magnet assembly from its inactive position to its active position. As described herein, the recovery magnet assembly can be advanced from its inactive position to its active position by movement of a recovery magnet assembly actuator.

[0159] When the retrieval magnet assembly is placed in the active position, it can attract barcode-containing beads disposed within the microwells. In some embodiments, the uniform magnetic force exerted by the retrieval magnet assembly on the barcode-containing beads can be sufficient to remove the barcode-containing beads from the microwells. When the barcode-containing beads are removed by the retrieval magnet assembly, the cells can remain within the microwells. In some embodiments, the magnetic force exerted by the retrieval magnet assembly on the barcode-containing beads can move the barcode-containing beads toward the top surface of the cartridge. The magnetic force exerted by the retrieval magnet assembly can maintain the beads above the microwells. While the beads are maintained above the microwells, they can be said to be retrieved by the retrieval magnet assembly.

[0160] Washing can be performed while the collection magnet assembly maintains the beads above the microwells. In some embodiments, a wash fluid can be introduced into the cartridge flow cell via an inlet. The wash fluid can flow through the flow cell to remove cells within the microwells. The cells can be deposited in a waste collection container aligned with the outlet of the flow cell. Washing the cells from the microwells allows for subsequent collection of only the beads previously placed in the microwells. In some embodiments, activation of the collection magnet assembly engages an interlock with the drawer, preventing movement of the drawer.

[0161] After the beads are retrieved by the retrieval magnet assembly, the beads can be collected in step 1305. In some embodiments, the retrieval magnet assembly is transitioned from its activated position to its deactivated position, releasing the beads from a maintained position above the microwells. As described herein, the retrieval magnet assembly can be transitioned from its activated position to its deactivated position by movement of a retrieval magnet assembly actuator. In some embodiments, upon release, the beads fall or return to the same microwells from which they were removed.

[0162] After the beads are released, the sample collection container in the sample collection holder can be aligned with the outlet of the cartridge. As described herein, the sample collection container in the sample collection holder can be aligned with the outlet by transitioning the actuator to a sample collection position. After alignment, fluid can be advanced through the flow cell to cause the beads to flow out the outlet and into the sample collection container. It may be desirable for the collection magnet assembly to be placed in its inactive position to allow for collection of the beads. After the beads are collected, workflow 1300 ends. In some embodiments, after collection of the beads, the sample collection container can be removed for further processing and / or analysis of the beads.

[0163] Library preparation In some cases, the method also includes a sequence library preparation protocol for further processing the analytes (e.g., DNA, RNA) obtained by the above steps to generate a sequence library (e.g., a cDNA library). In certain cases, the method includes creating a sequence-ready nucleic acid library from the processed sample. In certain versions, the sequence-ready nucleic acid library is sequenceable by using a next-generation sequencing (NGS) protocol.

[0164] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of targets. Creation of an indexed library is described, for example, in U.S. Pat. No. 10,676,779 and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference in their entireties. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) can differ from one another. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, in an indexed library of barcoded targets comprising a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide may differ from the labeled region of the second indexed polynucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or by a number or range of nucleotides between any two of these values, or by about these number of nucleotides, or by at least these number of nucleotides, or by at most these number of nucleotides. In some embodiments, generating an indexed library of barcoded targets comprises contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a labeled region, and performing first strand synthesis using reverse transcriptase to produce single-stranded, labeled cDNA molecules, each comprising a cDNA region and a labeled region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences.Generating an indexed library of barcoded targets can further include amplifying the single-stranded labeled cDNA molecules to create double-stranded labeled cDNA molecules, and performing nested PCR on the double-stranded labeled cDNA molecules to create labeled amplicons. In some embodiments, the method can include generating adapter-labeled amplicons.

[0165] Barcoding (e.g., stochastic barcoding) can involve using nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters can be added for sequencing, for example, using next-generation sequencing (NGS). Sequencing results can be used to determine the sequence of nucleotide fragments of one or more copies of cell labels, molecular labels, and targets.

[0166] Figure 14A shows a workflow for carrying out a particular embodiment of the method. As shown in step 1451, cells and beads are loaded into a cartridge of the invention. One cell is paired with one barcoded bead in a microwell. In step 1452, the cell is lysed and nucleic acid from the cell is hybridized to the beads. In step 1453, a collection magnet assembly is used to collect the beads. In step 1454, the nucleic acid is used to synthesize cDNA.

[0167] FIG. 14B is a schematic diagram illustrating a non-limiting exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), such as barcoded mRNAs or fragments thereof. In other words, FIG. 14B illustrates the exemplary cDNA synthesis step 1454 shown in FIG. 14A. As shown in step 1, the reverse transcription process can encode each mRNA molecule with a unique molecular label, a cellular label, and a universal PCR site. In particular, RNA molecules 1402 can be reverse transcribed to generate labeled cDNA molecules 1404 including cDNA regions 1406 by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 1410 to a poly(A) tail region 1408 of the RNA molecule 1402. Each of the barcodes 1410 can include a target binding region, e.g., a poly(dT) region 1412, a label region 1414 (e.g., a barcode sequence or molecule), and a universal PCR region 1416. In some embodiments, the cellular label can comprise 3 to 20 nucleotides. In some embodiments, the molecular label can comprise 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cellular label, wherein the universal label is the same for the plurality of stochastic barcodes on the solid support and the cellular label is the same for the plurality of stochastic barcodes on the solid support. In some embodiments, the universal label can comprise 3 to 20 nucleotides. In some embodiments, the cellular label comprises 3 to 20 nucleotides.

[0168] In some embodiments, label region 1414 can include a barcode sequence or molecular label 1418 and a cell label 1420. In some embodiments, label region 1414 can include one or more of a universal label, a dimensional label, and a cell label. Barcode sequence or molecular label 1418 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range between any of these values, or can be about, at least, or at most these values ​​in nucleotide length. The cell label 1420 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range of nucleotides in length between any of these values, or can be about, or can be at least, or can be at most these values ​​in nucleotides in length. The universal label can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range of nucleotides in length between any of these values, or can be about, or can be at least, or can be at most these values ​​in nucleotides in length. The universal label can be the same for multiple stochastic barcodes on a solid support, and the cell label is the same for multiple stochastic barcodes on a solid support. A dimension label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range between any of these values ​​in length, or may be about these values ​​in nucleotides in length, or may be at least these values ​​in nucleotides in length, or may be at most these values ​​in nucleotides in length.

[0169] In some embodiments, the number of different labels, such as barcode sequences or molecular labels 1418 and cellular labels 1420, included in label region 1414 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, or can be about these values, or can be at least these values, or can be at most these values. Each label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range of nucleotides in length between any of these values, or may be about, or may be at least, or may be at most, these values ​​in nucleotide length. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 , or may be a number or range between any of these values, or may be about these values, or may be at least these values, or may be at most these values. Additionally, each set of barcodes or stochastic barcodes 1410 can contain, for example, a unique labeled region 1414. The labeled cDNA molecules 1404 can be purified to remove excess barcodes or stochastic barcodes 1410. Purification can include Ampure bead purification.

[0170] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled into one tube and PCR amplified using a first pool of PCR primers and a first universal PCR primer. The unique label region 1414 enables pooling. In particular, the labeled cDNA molecules 1404 can be amplified to generate nested PCR labeled amplicons 1422. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the number of multiplex primers in a single reaction volume utilized for multiplex PCR amplification can be 10, 20, 40, 50, 70, 80, 90, 10, 20, 3 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 The amplification may include using a first PCR primer pool 1424 that includes custom primers 1426A-C that target specific genes and a universal primer 1428. The custom primers 1426A-C can hybridize to a region within the cDNA portion 1406' of the labeled cDNA molecule 1404. The universal primer 1428 can hybridize to the universal PCR region 1416 of the labeled cDNA molecule 1404.

[0171] As shown in step 3 of Figure 14B, the products from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the nested PCR labeled amplicon 1422 can be further amplified by nested PCR. Nested PCR can include multiplex PCR with a nested PCR primer pool 1430 of nested PCR primers 1432a-c and a second universal PCR primer 1428' in a single reaction volume. The number of different nested PCR primers 1430 included in the nested PCR primer pool can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, or can be about these values, or can be at least these values, or can be at most these values. Nested PCR primer 1432 includes adapter 1434 and can hybridize to a region within cDNA portion 1406" of labeled amplicon 1422. Universal primer 1428' includes adapter 1436 and can hybridize to universal PCR region 1416 of labeled amplicon 1422. Thus, step 3 creates adapter-labeled amplicon 1438. In some embodiments, nested PCR primer 1432 and second universal PCR primer 1428' may not include adapters 1434 and 1436. Instead, adapters 1434 and 1436 can be ligated to the product of the nested PCR to create adapter-labeled amplicon 1438.

[0172] As shown in step 4, the PCR product from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 1434 and 1436 can be used to perform one or more further assays on adapter-labeled amplicons 1438. Adapters 1434 and 1436 can hybridize to primers 1440 and 1442. One or more of primers 1440 and 1442 can be PCR amplification primers. One or more of primers 1440 and 1442 can be sequencing primers. One or more of adapters 1434 and 1436 can be used for further amplification of adapter-labeled amplicons 1438. One or more of adapters 1434 and 1436 can be used for sequencing of adapter-labeled amplicons 1438. The primers 1442 can include a plate index 1444 so that amplicons generated using the same set of barcodes or stochastic barcodes 1410 can be sequenced in a single sequencing reaction using next generation sequencing (NGS).

[0173] For example, as noted above, further details regarding library preparation workflows using systems of embodiments of the present invention can be found in U.S. Patent Nos. 9,598,736, 10,002,316, 10,527,171, 10,634,691, 10,676,779, 11,061,043, and 11,365,409, as well as U.S. Patent Application Publication No. 2016 / 0340720. and 2021 / 0171940, the disclosures of which are incorporated herein by reference in their entireties.

[0174] sample As described above, the method includes processing a sample. The sample can include one or more cells or nucleic acids from one or more cells. The sample can be a single cell or nucleic acids derived from a single cell. A sample for use in the disclosed methods can include one or more cells. In some embodiments, the plurality of cells can include one or more cell types. At least one of the one or more cell types can be brain cells, cardiac cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, circulating cells, or any combination thereof. In some embodiments, the cells are cancer cells excised from cancerous tissue, such as breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, melanoma, and non-melanoma skin cancer. In some embodiments, the cells are derived from a cancer but collected from a bodily fluid (e.g., circulating tumor cells). Non-limiting examples of cancers include adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, and fibrosarcoma. The sample can include a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof. The sample can include a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, or cells from a subject. The sample can be obtained from a human, a mammal, a dog, a rat, a mouse, a fish, a fly, an insect, a plant, a fungus, a bacterium, a virus, a vertebrate, or an invertebrate.

[0175] In some embodiments, the cell is infected with a virus and contains viral oligonucleotides. In some embodiments, the viral infection can be caused by a virus such as a single-stranded (positive-strand or "sense") DNA virus (e.g., parvovirus) or a double-stranded RNA virus (e.g., reovirus). In some embodiments, the cell is a bacterium. These can include either gram-positive or gram-negative bacteria. In some embodiments, the cell is a fungus. In some embodiments, the cell is a protozoan or other parasite.

[0176] As used herein, the term "cell" can refer to one or more cells. In some embodiments, the cells are normal cells, e.g., human cells at different stages of development, or human cells of different organ or tissue types. In some embodiments, the cells are non-human cells, e.g., other types of mammalian cells (e.g., mouse, rat, pig, dog, cow, and horse). In some embodiments, the cells are other types of animal or plant cells. In other embodiments, the cells can be any prokaryotic or eukaryotic cell.

[0177] In some embodiments, cells are sorted before associating the cells with the beads. For example, cells can be sorted by fluorescence-activated cell sorting or magnetic-activated cell sorting, or generally by flow cytometry. Cells can be filtered by size. In some embodiments, the retentate contains the cells associated with the beads. In some embodiments, the flow-through contains the cells associated with the beads. A sample can refer to a plurality of cells. A sample can refer to a monolayer of cells. A sample can refer to a thin section (e.g., a thin section of tissue). A sample can refer to a solid or semi-solid collection of cells, which can be arranged one-dimensionally on an array.

[0178] kit As noted above, aspects of the present invention further include kits. Kits of the present invention include a multi-microwell array flow cell cartridge of the present invention (e.g., as described above). In some cases, the kit includes a plurality of multi-microwell array flow cell cartridges. In some embodiments, the kit includes a sample collection container holder configured to receive a plurality of sample collection containers. According to some embodiments, the sample collection container holder includes a counterweight configured to maintain the sample collection container holder in an upright position. In some embodiments, the kit includes a plurality of sample collection containers. The kit may also include one or more waste collection containers.

[0179] In some cases, the components of the kit are provided in one or more sealable containers. In some cases, the one or more sealable containers are resealable, i.e., they may be opened and closed. Any convenient container may be employed, such as a pouch, bag, box, etc. In certain embodiments, a desiccant is provided in one or more containers, i.e., to control the moisture content within the container. Exemplary desiccants include silica gel, etc. In some cases, a multi-microwell-array-flow cell cartridge of the present invention can be used or partially used (e.g., by performing a method of the present invention on one or more fluid lanes of the cartridge) and placed in a sealable container for storage. A partially used multi-microwell-array-flow cell cartridge may be removed from the sealable container and used to prepare one or more additional samples, after which the cartridge may be returned to the container as needed. The sealable container may be configured to maintain the stability of a partially used cartridge so that it may be operable for subsequent use at one or more time points after first use, including at least 1 minute after first use, such as at least 5 minutes after first use, such as at least 30 minutes after first use, such as at least 1 hour after first use, such as at least 6 hours after first use, such as at least 1 day after first use, such as at least 1 week after first use, such as at least 2 weeks after first use, such as at least 1 month after first use, such as at least 3 months after first use, or at least 6 months after first use.

[0180] In some cases, the kit includes barcoded beads, e.g., the barcoded beads include a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain. In some such cases, the target capture domain is an oligo-dT sequence. In certain versions, the nucleic acid barcode further includes a unique molecular index (UMI). In some embodiments, the kit includes a stochastic barcode that cannot be attached to a bead. The kit can further include reagents such as a lysis buffer (such as those described above), a rinse / wash buffer, a hybridization buffer, and a reducing agent (e.g., dithiothreitol). In some embodiments, the kit further includes reagents (e.g., enzymes, primers, dNTPs, NTPs, RNAse inhibitors, or buffers) for performing a nucleic acid extension reaction, e.g., a reverse transcription reaction and a primer extension reaction. In some embodiments, the kit further includes reagents (e.g., enzymes, primers, dNTPs, NTPs, RNAse inhibitors, or buffers) for performing an amplification reaction to prepare a sequencing library. In some embodiments, the kit can include a ligase, a transposase, a reverse transcriptase, a DNA polymerase, an RNase, an exonuclease, or any combination thereof. In some embodiments, the kit includes reagents for homopolymeric tailing of molecules (e.g., a terminal transferase enzyme and dNTPs). The kit can include, for example, reagents for any of the enzymatic cleavage methods disclosed herein (e.g., ExoI nuclease, restriction enzymes). In some embodiments, the kit can include reagents for adapter ligation (e.g., a ligase enzyme, a reducing reagent). In some embodiments, the kit can include reagents for library preparation (e.g., adding sequencing library / flow cell primers), which can include sequencing / flow cell primers, enzymes for binding primers, dNTPs, etc.

[0181] Kit components may be present in separate containers, or multiple components may be present in a single container. For example, the template switch oligonucleotide and template switch polymerase may be provided in the same tube or in different tubes. In certain embodiments, it is often convenient to provide the components in lyophilized form, so that they are ready to use and can be conveniently stored at room temperature.

[0182] In addition to the above components, the kit may further include (in certain embodiments) instructions for practicing the method. These instructions may be present in the kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Another form in which these instructions may be present is as a computer-readable medium having the information recorded thereon, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote site.

[0183] Further details regarding aspects of the present invention can be found in U.S. Patent Nos. 9,598,736, 10,002,316, 10,527,171, 10,634,691, 10,676,779, 11,061,043, and 11,365,409, as well as U.S. Patent Application Publication Nos. 2016 / 0340720 and 2018 / 0276332. and 2021 / 0171940, the disclosures of which are incorporated herein by reference in their entireties.

[0184] Utilities For example, the systems, cartridges, methods, and kits of the present invention may be employed when it is desirable to prepare a biological sample for further analysis. For example, the present invention may be employed to prepare a biological sample for use in diagnostics, monitoring, and research purposes. In some cases, the systems, cartridges, methods, and kits of the present invention may be employed to increase the throughput of sample preparation and analysis by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, including 8-fold or more. In some cases, the present invention may be employed when it is desirable to simultaneously prepare and analyze multiple samples without experiencing batch effects. In some cases, the systems, cartridges, methods, and kits of the present invention may be used when there is a need to simplify the mechanism for sample preparation and reduce costs.

[0185] The following examples are offered by way of illustration and not by way of limitation.

[0186] experiment Example 1 Simulations were performed in COMSOL Multiphysics® to demonstrate the magnetic forces resulting from various arrangements of magnets in the retrieval magnet assembly. A simulation of the magnetic forces of four 3" x 1 / 2" x 1 / 8" magnets with alternating polarity is shown in Figure 15A. A simulation of the magnetic forces of four 3" x 1 / 2" x 1 / 8" magnets without alternating polarity is shown in Figure 15B. A simulation of one 3" x 3" x 1 / 8" magnet is shown in Figure 15C. As shown in Figures 15A-15C, the magnets with alternating polarity exhibit a uniform force.

[0187] Simulations were also performed to examine the effect of the distance between adjacent magnets on the magnetic force. Figure 15D shows various simulated magnet configurations. Figures 15E-15G show the results of the simulated configurations. A significant batch effect was observed in the configuration shown in Figure 15E.

[0188] A parametric comparison was performed for the different configurations shown in Figure 15D. As shown in Figure 15I, the "-2mm" and "aligned" magnet configurations have similar magnetic force field coverage across the centerline. The centerline is shown in Figure 15H. As shown in Figure 15J, the magnetic force is weakest along the centerline.

[0189] Example 2 The number of cells captured per lane of an 8-lane cartridge was measured. Over 40K cells per lane, or over 320K viable cells, were captured across the 8 lanes (Figure 16A). Capture rates of up to 80% were observed. Multiplexing rates were comparable to single-lane Express, and cell labeling collisions of less than 0.5% were achieved thanks to the high diversity of beads. No batch effect was observed. High gene expression correlation was observed with multi-lane and single-lane Rhapsody (Figure 16B).

[0190] Example 3 Flexible cell throughput per lane with the BD Rhapsody™ HT-Xpress was demonstrated, and the results are shown in Figures 17A-17D. All cell loading was performed on a single 8-lane cartridge, with 100, 1,000, 5,000, 10,000, 25,000, 40,000, 55,000, and 65,000 cells loaded simultaneously across lanes 1 through 8, respectively. This finding correlates with theoretical Poisson multiple loading for loading of 10,000 cells or more, meeting the criteria for cell capture efficiency and multiplexing rate.

[0191] Example 4 A single 8-lane cartridge was used partially over multiple days. Some of lanes 1-8 were used at one time, and the remaining lanes were used at different times for the same or different assays. On day 1, lanes 1 and 2 were used. On day 2, lanes 3, 4, and 5 were used. On day 3, lanes 6, 7, and 8 were used. The results are shown in Figures 18A-18C.

[0192] Example 5 The stability of partially used cartridges was studied for up to 4 months. Figures 19A-19B show the target + SMK assay in lanes 1 and 2 of an 8-lane cartridge using 1:1 Jurkat / Ramos cells on day 1. Figures 19C-19D show the WTA + Abseq assay in lanes 3 and 4 of an 8-lane cartridge using PBMCs. Day 1 and day 7 results on the various lanes show close correlation with the single-lane control.

[0193] Example 6 A 1:1:1 mixture of cells of various sizes was processed through the 8-lane cartridge. Cells of various sizes were captured with similar efficiency (80% capture rate at 20K loading). No batch effect was observed in samples captured on the single-lane and 8-lane cartridges. t-SNE analysis showed Jurkat / BT549 / K562 cell clusters (Figures 20A-B).

[0194] Example 7 The ability of the 8-lane cartridge to capture enriched neutrophils and sorted NK and T cells was evaluated compared to the single-lane cartridge. No batch effect was observed in samples captured with the single-lane and 8-lane cartridges. t-SNE analysis shows T cell / NK cell / neutrophil cell clusters (Figure 21A). Figure 21B shows the capture rates for various cell types.

[0195] Example 8 Twenty-four sample tubes of Jurkat / Ramos / THP1 cells were stained with the 24 flex SMK tags and mixed prior to the Rhapsody workflow. Target + SMK experiments were performed, demonstrating greater than 95% sensitivity and specificity for the 24 sample tags (Figures 22A-B). Two-step staining was employed, with anti-β2-microglobulin as the primary antibody (Figures 22C-D).

[0196] Example 9 Figure 23A shows the workflow for characterizing full-length T cell receptor sequences, along with the transcriptome and surface proteins of antigen-specific T cells. Good resolution between Abseq (CITE-seq) and dCODE reads is shown in Figure 23B. As shown in Figure 23C, TCR alpha / beta chain clonotypes were found in clusters of cells with high detection of the specific antigen CMV3 dCODE.

[0197] Example 10 Simultaneous profiling of mRNA, surface, and intracellular proteins was performed. High correlation between mRNA and surface Abseq + / - intracellular Abseq is shown in Figures 24A-24D. Detection of pSTAT1, pSTAT5, and pSTAT6 by intracellular CITE-seq is shown in Figures 24E-24L. There is agreement between intracellular CITE-seq and flow cytometry. In CD4+ T cells, phosphorylation was observed after 15 minutes of stimulation with hIFN-α, hIL-2, and hIL-4.

[0198] Example 11 As shown in Figures 25A-25C, there is no batch effect between technical replicates between the two cartridges loaded with tagged cells. The markers divide the sample into six distinct populations, and the results of the experiment demonstrate the maintenance of independence of cell populations for each cartridge and lane when overlaid on a t-SNE plot illustrating the tagged cell clusters of the sample.

[0199] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following clauses.

[0200] 1. A tray configured to receive a multi-microwell array flow cell cartridge; a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the cartridge when in an activated position; A system comprising:

[0201] 2. The system of clause 1, wherein the retrieval magnet assembly is configured to apply a uniform magnetic force from a position above the tray.

[0202] 3. The system of clause 1 or 2, wherein the uniform magnetic force is a magnetic field in the range of 650 Gauss to 1325 Gauss.

[0203] 4. The system of any one of the preceding clauses, wherein the retrieval magnet assembly is configured to apply a uniform magnetic force via multiple magnets.

[0204] 5. The system of clause 4, wherein the plurality of magnets have alternating polarities.

[0205] 6. The system of clause 4 or 5, wherein the number of magnets in the recovery magnet assembly is in the range of 2 to 10.

[0206] 7. The system of clause 6, wherein the recovery magnet assembly comprises four magnets.

[0207] 8. The system of any one of clauses 4 to 7, wherein the plurality of magnets includes rare earth magnets.

[0208] 9. The system of clause 8, wherein the rare earth magnet is a neodymium magnet.

[0209] 10. The system of clause 8, wherein the rare earth magnet is a samarium-cobalt magnet.

[0210] 11. A system according to any one of the preceding clauses, wherein the plurality of magnets are bar magnets.

[0211] 12. The system of any one of clauses 4 to 11, wherein the plurality of magnets are ring magnets.

[0212] 13. The system of clause 12, wherein the plurality of magnets are arranged in a bull's-eye configuration.

[0213] 14. A system according to any one of the preceding clauses, wherein the retrieval magnet assembly is operable between an operative position in which the retrieval magnet assembly is positioned adjacent to the tray and a non-operative position in which the retrieval magnet is positioned further away from the tray relative to the operative position.

[0214] 15. The system of any one of the preceding clauses, further comprising a sample collection container holder configured to receive a plurality of sample collection containers for collecting analytes from the cartridge.

[0215] 16. The system of clause 15, further comprising a plurality of sample collection containers.

[0216] 17. The system of clause 16, wherein the system comprises a number of sample collection containers in the range of 2 to 10.

[0217] 18. A system according to clause 16 or 17, wherein the system comprises a number of sample collection containers corresponding to the number of flow cells in the cartridge.

[0218] 19. The system of clause 17 or 18, wherein the system comprises eight sample collection containers.

[0219] 20. The system of any one of clauses 15 to 19, wherein the sample collection container holder comprises a counterweight configured to maintain the sample collection holder in an upright position.

[0220] 21. A system according to any one of clauses 15 to 20, wherein the system and the sample collection container holder have complementary shapes such that the sample collection container holder can be received in the system in a single orientation.

[0221] 22. A system according to any one of the preceding clauses, further comprising a drawer that is movable between a plurality of different positions within the system.

[0222] 23. The system of any one of the preceding clauses, further comprising a waste collection container for collecting liquid waste from the multi-microwell-array-flow cell cartridge.

[0223] 24. A system as described in clause 23, comprising a single waste collection container.

[0224] 25. A system according to any one of clauses 23 to 24, further comprising an interlock configured to prevent collection of sample liquid into the waste collection container when the recovery magnet assembly is in the activated position.

[0225] 26. The system of any one of the preceding clauses, wherein the tray comprises latches for holding the multi-microwell-array-flow cell cartridge.

[0226] 27. A system according to any one of the preceding clauses, wherein the system does not include a dissolving magnet at a position below the tray.

[0227] 28. A multi-microwell array flow cell cartridge further comprising: a multi-microwell array flow cell cartridge having a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid 10. The system of any one of the preceding clauses, comprising:

[0228] 29. The system of clause 28, wherein the multi-microwell-array-flow cell cartridge comprises a number of fluid lanes ranging from 2 to 10.

[0229] 30. The system of clause 29, wherein the multi-microwell-array-flow cell cartridge comprises eight fluid lanes.

[0230] 31. A system according to any one of clauses 28 to 30, wherein each outlet is stepped to prevent siphoning of liquid from the flow cell.

[0231] 32. A system according to any one of clauses 28 to 31, wherein each flow cell comprises an elongated channel.

[0232] 33. The system of clause 32, wherein the elongated channel is in the range of 50 mm to 100 mm in length.

[0233] 34. The system of any one of clauses 28 to 33, wherein each microwell array comprises between 250,000 microwells and 300,000 microwells.

[0234] 35. Each microwell array has 36,000 microwells / cm 2 ~42,000 microwells / cm 2 35. The system of any one of clauses 28 to 34, comprising a density in the range of

[0235] 36. A system described in any one of clauses 28 to 35, wherein the tray has a shape complementary to the shape of the multi-microwell-array-flow cell cartridge so that the multi-microwell-array-flow cell cartridge can be received in the tray in a single orientation.

[0236] 37. The system of clause 36, wherein the multi-microwell-array-flow cell cartridge comprises chamfered corners.

[0237] 38. A system according to any one of the preceding clauses, further comprising a drip receptacle positioned below the tray and configured to receive liquid expelled from the cartridge.

[0238] 39. The system of clause 38, wherein the drip receptacle is detachable.

[0239] 40. A method for processing a cell sample, the method comprising: a tray configured to receive a multi-microwell-array-flow cell cartridge; a retrieval magnet assembly configured to apply a uniform magnetic force to a flow cell of the multi-microwell array flow cell cartridge when in an active position; introducing a multi-microwell-array-flow cell cartridge into a system comprising: Loading a sample into a multi-microwell array flow cell cartridge; actuating the retrieval magnet assembly to an active position and applying a uniform magnetic force to the sample within the multi-microwell array flow cell cartridge to produce a processed sample; Collecting the processed sample from the multi-microwell array flow cell cartridge; A method comprising:

[0240] 41. The method of clause 40, wherein the method comprises loading a plurality of different samples into different flow cells of the cartridge.

[0241] 42. The method according to clause 41, wherein the number of different samples is in the range of 2 to 10.

[0242] 43. The method of any one of clauses 40 to 42, further comprising loading the multi-well cartridge with a lysis buffer after loading the sample liquid into the multi-well cartridge.

[0243] 44. The method of clause 43, wherein the method comprises filling the multi-well cartridge with lysis buffer without the use of a lysis magnet.

[0244] 45. The method of any one of clauses 40 to 44, wherein the method includes loading barcoded beads into a multi-well cartridge prior to actuation of the retrieval magnet assembly.

[0245] 46. ​​The method of clause 45, wherein the barcoded bead comprises a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain.

[0246] 47. The method of clause 46, wherein the target capture domain is a poly(T) sequence.

[0247] 48. The method of clause 46, wherein the nucleic acid barcode further comprises a unique molecular index (UMI).

[0248] 49. The method of any one of clauses 40 to 48, further comprising applying a uniform magnetic field from a position above the tray.

[0249] 50. The method of any one of clauses 40 to 49, wherein the recovery magnet assembly, when in the active position, is configured to apply a uniform magnetic force to the flow cells of the multi-microwell-array-flow cell cartridge via the plurality of magnets.

[0250] 51. The method of clause 50, wherein the plurality of magnets have alternating polarities.

[0251] 52. The method of clause 50 or 51, wherein the number of magnets in the recovery magnet assembly is in the range of 2 to 10.

[0252] 53. The method of clause 52, wherein the retrieval magnet assembly comprises four magnets.

[0253] 54. The method of any one of clauses 50 to 53, wherein the plurality of magnets comprises rare earth magnets.

[0254] 55. The method of any one of clauses 40 to 54, wherein the system comprises a sample collection container holder configured to receive a plurality of sample collection containers for collecting analytes from the sample in the cartridge.

[0255] 56. The method of clause 55, wherein the method includes collecting the processed samples in a plurality of sample collection containers.

[0256] 57. The method of clause 56, wherein the method includes collecting the processed sample in a number of sample collection containers in the range of 2 to 10.

[0257] 58. The method of clause 56 or 57, wherein the method comprises collecting the processed sample in a number of sample collection vessels corresponding to the number of flow cells in the cartridge.

[0258] 59. The method of clause 57 or 58, wherein the method includes collecting eight processed samples in eight sample collection containers.

[0259] 60. The method of any one of clauses 55 to 59, wherein the sample collection container holder comprises a counterweight configured to maintain the sample collection holder in an upright position.

[0260] 61. A method according to any one of clauses 40 to 60, wherein the system comprises a drawer that is movable between a plurality of different positions within the system.

[0261] 62. The method of clause 61, further comprising moving the drawer between a plurality of different locations within the system.

[0262] 63. The method of any one of clauses 40 to 62, further comprising collecting liquid waste from the multi-microwell-array-flow cell cartridge in a waste collection container.

[0263] 64. A method according to any one of clauses 61 to 63, wherein the system comprises an interlock configured to prevent collection of sample liquid into the waste collection container when the recovery magnet assembly is in the activated position.

[0264] 65. The method of clause 62, further comprising engaging an interlock during operation of the retrieval magnet assembly.

[0265] 66. A multi-microwell array flow cell cartridge, a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid 66. The method of any one of clauses 40 to 65, comprising:

[0266] 67. The method of clause 66, wherein the cartridge comprises a number of fluid lanes in the range of 2 to 10.

[0267] 68. The method of clause 67, wherein the cartridge comprises eight fluid lanes.

[0268] 69. A method according to any one of clauses 66 to 68, wherein the outlet is stepped to prevent siphoning of liquid from the flow cell.

[0269] 70. The method of any one of clauses 66 to 69, wherein each flow cell comprises an elongated channel.

[0270] 71. The method of clause 70, wherein the elongated channel is in the range of 50mm to 100mm in length.

[0271] 72. The method of any one of clauses 66 to 71, wherein each microwell array comprises between 250,000 microwells and 300,000 microwells.

[0272] 73. Each microwell array has 36,000 microwells / cm 2 ~42,000 microwells / cm 2 73. The method of any one of clauses 66 to 72, comprising a density in the range of

[0273] 74. The method of any one of clauses 40 to 73, wherein the method further comprises producing a sequence-ready nucleic acid library from the processed sample.

[0274] 75. The method according to clause 74, wherein the sequence-ready nucleic acid library is sequenceable by using a next-generation sequencing protocol.

[0275] 76. The method of any one of clauses 40 to 75, wherein the method is a method of genomic analysis.

[0276] 77. The method of any one of clauses 40 to 75, wherein the method is a method of epigenomic analysis.

[0277] 78. The method of any one of clauses 40 to 75, wherein the method is a method of transcriptome analysis.

[0278] 79. The method of any one of clauses 40 to 75, wherein the method is a method of proteome analysis.

[0279] 80. The method of any one of clauses 40 to 75, wherein the method is a method of multi-omic analysis.

[0280] 81. The method of clause 80, wherein the multi-omic analysis includes at least transcriptome analysis and proteome analysis.

[0281] 82. A plurality of fluid lanes, each of which: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid A multi-microwell array flow cell cartridge comprising:

[0282] 83. The multi-microwell-array-flow cell cartridge of clause 82, wherein the multi-microwell-array-flow cell cartridge comprises a number of fluid lanes in the range of 2 to 10.

[0283] 84. The multi-microwell-array-flow cell cartridge of clause 83, wherein the multi-microwell-array-flow cell cartridge comprises eight fluid lanes.

[0284] 85. A multi-microwell-array-flow cell cartridge according to any one of clauses 82 to 84, wherein the outlet is stepped to prevent siphoning of liquid from the flow cell.

[0285] 86. The multi-microwell-array-flow cell cartridge of any one of clauses 82 to 85, wherein the outlet is a tapered conical orifice.

[0286] 87. The multi-microwell-array-flow cell cartridge of clause 86, wherein the tapered conical orifice is configured to induce droplet formation for channel flow rates between 20 μL / s and 500 μL / s.

[0287] 88. The multi-microwell-array-flow cell cartridge of any one of clauses 82 to 87, wherein each flow cell comprises an elongated channel.

[0288] 89. The multi-microwell-array-flow cell cartridge of clause 88, wherein the elongated channel ranges in length from 50 mm to 100 mm.

[0289] 90. The multi-microwell-array-flow cell cartridge of any one of clauses 82 to 89, wherein each microwell array comprises between 250,000 and 300,000 microwells.

[0290] 91. Each microwell array has 36,000 microwells / cm 2 ~42,000 microwells / cm 2 91. The multi-microwell array flow cell cartridge of any one of clauses 82 to 90, comprising a density in the range of:

[0291] 92. The multi-microwell-array-flow cell cartridge of any one of clauses 82 to 91, wherein the multi-microwell-array-flow cell cartridge comprises chamfered corners.

[0292] 93. The multi-microwell array flow cell cartridge of any one of clauses 82 to 92, wherein each inlet is provided with a gasket.

[0293] 94. The multi-microwell-array-flow cell cartridge of clause 93, wherein the gasket has a Shore durometer in the range of 10 to 80.

[0294] 95. The multi-microwell-array-flow cell cartridge of clause 93, wherein the gasket is configured to taper lock onto the pipette tip.

[0295] 96. The multi-microwell array flow cell cartridge of clause 95, wherein the taper lock has a Z-axis tolerance of 0.5 mm.

[0296] 97. A sample vessel holder configured to receive a plurality of sample collection vessels for collecting analytes from the multi-microwell-array-flow cell cartridge of any one of clauses 82 to 96.

[0297] 98. The sample vessel holder of clause 97, wherein the sample vessel holder is configured to receive a number of sample collection vessels in the range of 2 to 10.

[0298] 99. The sample vessel holder of clause 98, wherein the sample vessel holder is configured to receive eight sample collection vessels.

[0299] 100. The sample container holder of any one of clauses 97 to 99, wherein the sample collection container holder comprises a counterweight configured to maintain the sample collection holder in an upright position.

[0300] 101. A sample container holder according to any one of clauses 97 to 100, wherein the sample collection container holder has a shape complementary to the cellular analysis system such that the sample collection container holder can be received in the cellular analysis system in a single orientation.

[0301] 102. A multi-microwell array flow cell cartridge having a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid A kit comprising:

[0302] 103. The kit of clause 102, wherein the cartridge comprises a number of fluid lanes in the range of 2 to 10.

[0303] 104. The kit of clause 103, wherein the cartridge comprises eight fluid lanes.

[0304] 105. A kit according to any one of clauses 102 to 104, wherein each outlet is stepped to prevent siphoning of liquid from the flow cell.

[0305] 106. A kit according to any one of clauses 102 to 105, wherein the outlet is a tapered conical orifice.

[0306] 107. The kit of clause 106, wherein the tapered conical orifice is configured to induce droplet formation for a channel flow rate of 20 μL / s to 500 μL / s.

[0307] 108. The kit of any one of clauses 102 to 107, wherein each flow cell comprises an elongated channel.

[0308] 109. The kit of clause 108, wherein the elongated channel is in the range of 50 mm to 100 mm in length.

[0309] 110. The kit of any one of clauses 102 to 109, wherein each microwell array comprises between 250,000 microwells and 300,000 microwells.

[0310] 111. Each microwell array has 36,000 microwells / cm 2 ~42,000 microwells / cm 2 111. The kit of any one of clauses 102 to 110, comprising a density in the range of

[0311] 112. The kit of any one of clauses 102 to 111, wherein the multi-microwell array flow cell cartridge has chamfered corners.

[0312] 113. A kit according to any one of clauses 102 to 112, wherein each inlet is provided with a gasket.

[0313] 114. The kit of clause 113, wherein the gasket has a Shore durometer in the range of 10 to 80.

[0314] 115. The kit of clause 113, wherein the gasket is configured to taper lock onto the pipette tip.

[0315] 116. The kit of clause 113, wherein the taper lock has a Z-axis tolerance of 0.5 mm.

[0316] 117. The kit of any one of clauses 102 to 116, wherein the kit comprises a plurality of multi-microwell-array-flow cell cartridges.

[0317] 118. The kit of any one of clauses 102 to 117, further comprising a sample collection container holder configured to receive a plurality of sample collection containers.

[0318] 119. The kit of clause 118, wherein the specimen collection container holder comprises a counterweight configured to maintain the specimen collection container holder in an upright position.

[0319] 120. The kit of any one of clauses 102 to 119, further comprising a plurality of sample collection containers.

[0320] 121. The kit of any one of clauses 102 to 120, further comprising a waste collection container.

[0321] 122. The kit of any one of clauses 102 to 121, further comprising a cell lysis buffer.

[0322] 123. The kit of any one of clauses 102 to 122, further comprising a hybridization buffer.

[0323] 124. The kit of any one of clauses 102 to 123, further comprising a wash buffer.

[0324] 125. The kit of any one of clauses 102 to 124, further comprising a reducing agent.

[0325] 126. The kit of any one of clauses 102 to 125, further comprising barcoded beads.

[0326] 127. The kit of clause 126, wherein the barcoded bead comprises a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain, and a target capture domain.

[0327] 128. The kit of clause 127, wherein the target capture domain is an oligo dT sequence.

[0328] 129. The kit of clause 127, wherein the nucleic acid barcode further comprises a unique molecular index (UMI).

[0329] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0330] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts with which the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a dedication to the public, regardless of whether such disclosure is expressly recited in the claims.

[0331] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a limitation in a claim only when the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of such limitation in the claim; if such precise phrases are not used in a claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0332] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing dates of U.S. Provisional Patent Application No. 63 / 468,622, filed May 24, 2023, and U.S. Provisional Patent Application No. 63 / 443,326, filed February 3, 2023, the disclosures of which are incorporated herein by reference.

Claims

1. a tray configured to receive a multi-microwell-array-flow cell cartridge; a retrieval magnet assembly configured to apply a uniform magnetic force to a flow cell of the cartridge when in an activated position; A system comprising:

2. The system of claim 1 , wherein the retrieval magnet assembly is configured to apply the uniform magnetic force from a position above the tray.

3. The system of claim 1 or 2, wherein the retrieval magnet assembly is configured to apply the uniform magnetic force via multiple magnets.

4. 4. The system of claim 1, wherein the retrieval magnet assembly is operable between an operative position in which the retrieval magnet assembly is positioned adjacent to the tray and a non-operative position in which the retrieval magnet is positioned further away from the tray relative to the operative position.

5. The system of claim 1 , further comprising a sample collection container holder configured to receive a plurality of sample collection containers for collecting analytes from the cartridge.

6. The system of claim 1 , further comprising a drawer that is movable between a plurality of different positions within the system.

7. 7. The system of claim 1, further comprising a waste collection container for collecting liquid waste from the multi-microwell-array-flow cell cartridge.

8. 8. The system of claim 1, wherein the tray comprises latches for holding the multi-microwell-array-flow cell cartridge.

9. The system of claim 1 , wherein the system does not include a dissolving magnet below the tray.

10. The multi-microwell-array-flow cell cartridge further comprises a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid The system of claim 1 , comprising:

11. 11. The system of claim 1, further comprising a drip receptacle positioned below the tray and configured to receive liquid expelled from the cartridge.

12. A method for processing a cell sample, said method comprising: a tray configured to receive a multi-microwell-array-flow cell cartridge; and a retrieval magnet assembly configured to apply a uniform magnetic force to a flow cell of the multi-microwell-array-flow cell cartridge when in an active position; introducing the multi-microwell array flow cell cartridge into a system comprising: loading the multi-microwell array flow cell cartridge with a sample; actuating the retrieval magnet assembly to the operating position and applying the uniform magnetic force to the sample in the multi-microwell-array-flow cell cartridge to produce a processed sample; collecting the processed sample from the multi-microwell array flow cell cartridge; A method comprising:

13. a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid A multi-microwell array flow cell cartridge comprising:

14. 13. A sample vessel holder configured to receive a plurality of sample collection vessels for collecting analytes from the multi-microwell-array-flow cell cartridge of claim 12.

15. a multi-microwell-array-flow cell cartridge having a plurality of fluid lanes, each fluid lane comprising: an inlet for receiving a liquid; a flow cell comprising a microwell array; An outlet for discharging the liquid A kit comprising:

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