System for processing cell samples, microporous array flow cell cartridges used therein, and methods of using same

Abstract

A system for processing a cell sample is provided. An embodiment of a system includes a tray configured to receive a microporous 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 the activated position. Methods are also provided that may include introducing a microporous array flow cell cartridge into a system of the invention, loading a sample into the microporous array flow cell cartridge, driving a retrieval magnet assembly to an enabled position and applying a uniform magnetic force to the sample within the microporous array flow cell cartridge to produce a processed sample; and collecting the treated sample from the microporous array flow cell cartridge. In addition, a microporous array flow cell cartridge is disclosed. Embodiments of the subject cartridge include a plurality of fluid lanes, each including an inlet for receiving a liquid, a flow cell including an array of micropores, and an outlet for discharging the liquid.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 63 / 468,622, filed on May 24, 2023, and to U.S. Provisional Patent Application Serial No. 63 / 443,326, filed on February 3, 2023, the disclosures of which are incorporated herein by reference. Background Art

[0003] The ability to detect and quantify specific nucleic acid and protein molecules in individual cells is crucial for understanding the role of cellular diversity in development, health, and disease. Flow cytometry has become a standard technique for high-throughput detection of protein markers on single cells and has been widely used in basic research and clinical diagnostics. In contrast, nucleic acid measurements (such as mRNA expression) are typically performed on large numbers of samples, thereby masking the contributions from individual cells.

[0004] Methods and techniques such as stochastic barcoding can be used for cellular analysis. For example, stochastic barcoding can be used to decipher cellular physiology, such as the protein and / or gene expression profile of a single cell, thereby determining its state using techniques such as reverse transcription, polymerase chain reaction (PCR) amplification, and next-generation sequencing (NGS). However, the detected expression profile can be associated with two or more different cell types, which can distort the interpretation of the expression profile.

[0005] Array technology has been used in biomedical research. The array is equipped with probes that can hybridize with target molecules with labels (e.g., fluorescence). The feature on the array is a small cluster of identical or similar probes that have a specific molecular sequence, such as DNA or RNA. Identifying the labeling pattern on the hybridization array can infer the hybridization that occurred in the sample, thereby further assisting biomedical research. U.S. Patent Nos. 10,634,691 and 11,061,043 describe loading stations for loading and recovering particles in array devices; the disclosures of these patents are incorporated herein by reference in their entirety.

[0006] To characterize the complexity of cellular systems, there is an urgent need to develop methods, devices, and systems for monitoring gene expression in large numbers across thousands of cells. Current technologies can measure gene expression in single cells in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells, while each cell is co-localized with barcoding reagent beads in a compartment. Summary of the Invention

[0007] The inventors have recognized that there continues to be a need for improved systems and methods for preparing sequencing-ready nucleic acid libraries from single cells. For example, while the loading stations described in U.S. Pat. Nos. 10,634,691 and 11,061,043 have improved the process of preparing cell samples, further improvements have been found to be needed. Embodiments of the present invention address these and other needs.

[0008] Aspects of the present invention include systems for processing cell samples. The system of an embodiment of the present invention includes a tray configured to receive a multi-microwell array flow cell box; and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the box when in an enabled position. In some 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 some cases, the uniform magnetic force can be a magnetic field in the range of 650 gauss to 1325 gauss. In some cases, the retrieval magnet assembly is configured to apply the uniform magnetic force via a plurality of magnets. In some such cases, the plurality of magnets have alternating polarity. The number of magnets in the retrieval magnet assembly can vary, and in some cases ranges from 2 to 10 (e.g., 4). The type of magnet in the retrieval magnet assembly can also vary. In some cases, the magnet is a rare earth magnet (e.g., a neodymium magnet and / or a samarium cobalt magnet). The shape of the magnet can also vary. In some cases, the multiple magnets are bar magnets. In other cases, the multiple magnets are ring magnets. In some such cases, the magnets in the multiple magnets are arranged in a bull's-eye configuration. The system of the present invention can be configured to adjust the position of the retrieval magnet assembly to process the cell sample. In some embodiments, the retrieval magnet assembly is drivable between the enabled position and the disabled position, wherein the enabled position is where the retrieval magnet assembly is located near the tray, and the disabled position is where the retrieval magnet is located at a greater distance from the tray relative to the enabled position.

[0009] In some forms, the system includes a sample collection container support, which is configured to receive a plurality of sample collection containers for collecting analytes from the box. In some such forms, the sample collection container support includes a counterweight, which is configured to maintain the sample collection container in an upright position. The system and the sample collection container support can also have complementary shapes so that the sample collection container support can be received in the system with a single orientation. In some aspects, the system also includes a plurality of sample collection containers. The number of sample collection containers can vary, and in some cases the range can be 2 to 10 (e.g., 8). In some cases, the system includes a sample collection container whose number matches the number of flow cells in the box. In some embodiments, a waste collection container is used to collect liquid waste from the multi-microporous array flow cell box. In some cases, the system includes an interlocking device, which is configured to prevent sample liquid from being collected in the waste collection container when the retrieval magnet assembly is in the enabled position. In some cases, the tray includes a latch for holding the multi-microporous array flow cell box. In selected forms, the system does not include a lysis magnet positioned below the tray.

[0010] In some cases, the system additionally includes the multi-micropore array flow cell box. The multi-micropore array flow cell box of interest includes multiple fluid swimming lanes, each fluid swimming lane including an inlet for receiving liquid, a flow cell including a micropore array, and an outlet for discharging liquid. In some cases, the multi-micropore array flow cell box includes a number range of 2 to 10 (e.g., 8) fluid swimming lanes. In some forms, each outlet is stepped to prevent siphoning liquid from the flow cell. In some embodiments, each flow cell includes an elongated channel. The length of the elongated channel can vary, and in some cases the range can be 50 mm to 100 mm. The number of micropores in each micropore array can also vary, and in some cases the range can be 250,000 micropores to 300,000 micropores. The micropore density in each micropore can vary, and in some cases the range can be 36,000 micropores / cm2 to 42,000 micropores / cm2.

[0011] In some cases, the tray has a shape that is complementary to the shape of the multi-microwell array flow cell cassette, such that the multi-microwell array flow cell cassette can be received in the tray in a single orientation. In some such cases, the multi-microwell array flow cell cassette comprises chamfered corners. In an embodiment, the system additionally comprises a drip vessel positioned below the tray, the drip vessel being configured to receive liquid discharged from the cassette.

[0012] Aspects of the present invention also include methods for processing cell samples. The method of interest includes introducing a multi-microporous array flow cell box (e.g., as described above) into a system of the present invention (e.g., as described above). The system of interest for the subject method includes a tray configured to receive a multi-microporous array flow cell box; and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the multi-microporous array flow cell box when in an enabled position. The method also includes loading the sample into the multi-microporous array flow cell box, driving the retrieval magnet assembly to the enabled position and applying the uniform magnetic force to the sample in the multi-microporous array flow cell box to produce a processed sample, and collecting the processed sample from the multi-microporous array flow cell box.

[0013] In some cases, the method includes loading a plurality of different samples into different flow cells of the box. The number of samples can vary, and in some cases can range from 2 to 10 (e.g., 8). The method according to certain embodiments includes loading the lysis buffer into the porous box after the sample liquid is loaded into the porous box. In some forms, the method includes loading the lysis buffer into the porous box without using a lysis magnet. The method according to the selected form of the present invention includes loading the barcoded beads into the porous box before the drive of the retrieval magnet assembly. In some cases, the barcoded beads can include a nucleic acid barcode, which includes a universal primer binding domain, a cell marker domain, and a target capture domain. In the selected embodiment, the target capture domain is a poly (T) sequence. In some cases, the nucleic acid barcode also includes a unique molecular index (UMI).

[0014] The type of analysis performed in the subject method may vary as needed. In some cases, the method comprises generating a sequencing-ready nucleic acid library from the processed sample. In certain embodiments, the sequencing-ready nucleic acid library is sequenceable using a next-generation sequencing protocol. In further embodiments, the method is a method of genomic analysis, epigenomic analysis, transcriptomic analysis, or proteomic analysis. In selected forms, the method is a multi-omic analysis method, for example, wherein the multi-omic analysis comprises at least transcriptomic and proteomic analysis.

[0015] The methods of the present invention also include a multi-micropore array flow cell box. The box of interest contains a plurality of fluid lanes, each fluid lane comprising an inlet for receiving a liquid, a flow cell comprising a micropore array, and an outlet for discharging the liquid. The disclosed multi-micropore array flow cell box is configured for use with the subject systems and methods. In addition, aspects of the present invention include a sample container holder configured to receive a plurality of sample collection containers for collecting analytes from the multi-micropore array flow cell box of the present invention. In some cases, the sample collection container holder has a shape complementary to the cell analysis system such that the sample collection container holder can be received in the cell analysis system in a single orientation.

[0016] Aspects of the present invention may further include a test kit. The test kit of the present invention includes a multi-microwell array flow cell box of the present invention. As discussed above, the multi-microwell array flow cell box of the present invention comprises a plurality of fluid lanes, each fluid lane comprising an inlet for receiving a liquid, a flow cell comprising a microwell array, and an outlet for discharging the liquid. The test kit may also include one or more instruments for implementing the method of the present invention. For example, the test kit according to some embodiments comprises a sample collection container holder, one or more sample collection containers, and one or more waste collection containers. In some cases, the test kit further comprises one or more reagents for implementing the method of the present invention. For example, the test kit according to some embodiments may comprise a hybridization buffer, a wash buffer, a reducing agent, and barcoded beads. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] This patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication containing these drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] The present invention may be better understood by reading the following detailed description in conjunction with the accompanying drawings, which include the following figures:

[0019] Figures 1A-1E Depicted are a retrieval magnet assembly and tray according to certain embodiments, and the retrieval magnet assembly from a deactivated position ( Figures 1A-1B ) to the enabled position ( Figures 1C-1D ) driver.

[0020] Figures 2A-2B Depicted are retrieval magnet assemblies according to certain embodiments.

[0021] Figures 3A-3H Depicted are systems for processing cell samples, according to certain embodiments.

[0022] Figures 4A-4B A travel tray according to certain embodiments is depicted.

[0023] Figures 5A-5B An interlock according to certain embodiments is depicted.

[0024] Figures 6A-6C Shown are multi-microwell array flow cell cartridges according to certain embodiments.

[0025] Figures 7A-7H Shown are different views of a multi-microwell array flow cell cartridge according to certain embodiments.

[0026] Figure 8 Depicted are alignment features of a multi-microwell array flow cell cartridge according to certain embodiments.

[0027] Figures 9A-9C Different depictions of the microwells of the in-cartridge flow cell of the present invention are shown.

[0028] Figure 10 Depicted is fluid flow through a multi-microwell array flow cell cartridge, according to certain embodiments.

[0029] Figures 11A-11H Depicted are cartridge carriers according to certain embodiments.

[0030] Figures 12A-12C A sample collection container holder according to certain embodiments is depicted.

[0031] Figure 13 Flowcharts for practicing the methods of the present invention are presented, according to certain embodiments.

[0032] Figure 14A Flowcharts for practicing the methods of the present invention are presented, according to certain embodiments.

[0033] Figure 14B is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of barcoded targets (eg, randomly barcoded targets), such as barcoded mRNAs or fragments thereof.

[0034] Figures 15A-15J Experimental data on retrieving the magnetic flux density of magnets in a magnet assembly are presented.

[0035] Figures 16A-16B Showcases BD Rhapsody TM HT-Xpress for higher throughput data.

[0036] Figures 17A-17D Demonstrated the use of BD Rhapsody TM Flexible cell throughput data per channel for HT-Xpress.

[0037] Figures 18A-18C Demonstrates some use of BD RhapsodyTM Flexible throughput data for the HT-Xpress 8-lane cassette.

[0038] Figures 19A-19D Demonstrates some use of BD Rhapsody TM Flexible throughput data for the HT-Xpress 8-lane cassette.

[0039] Figures 20A-20B Demonstrated the use of BD Rhapsody TM HT-Xpress captures data on cells of different sizes.

[0040] Figures 21A-21B Demonstrated the use of BD Rhapsody TM HT-Xpress captures data on fragile cells.

[0041] Figures 22A-22D Data related to two-step staining are shown.

[0042] Figures 23A-23C Shows the use of BD Rhapsody TM Antigen-specific T cell profiling by single-cell analysis. Figure 23C From top to bottom it contains SEQ ID NOs: 3-13.

[0043] Figures 24A-24L Simultaneous profiling of mRNA, surface proteins, and intracellular proteins is shown.

[0044] Figures 25A-25C This indicates that there is no batch effect between technical replicates. DETAILED DESCRIPTION

[0045] A system for processing a cell sample is provided. The system of an embodiment of the present invention comprises a tray configured to receive a multi-micropore array flow cell cassette; and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the cassette when in an enabled position. A method for practicing the present invention is also provided, wherein an embodiment comprises introducing a multi-micropore array flow cell cassette into the system of the present invention, loading a sample into the multi-micropore array flow cell cassette, driving the retrieval magnet assembly to an enabled position and applying a uniform magnetic force to the sample within the multi-micropore array flow cell cassette to produce a processed sample; and collecting the processed sample from the multi-micropore array flow cell cassette. In addition, a multi-micropore array flow cell cassette is disclosed. The subject cassette comprises a plurality of fluid lanes, each fluid lane comprising an inlet for receiving a liquid, a flow cell comprising a micropore array, and an outlet for discharging the liquid.

[0046] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0047] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range (unless the context clearly dictates otherwise, to the tenth of the unit of the lower limit) and any other stated or intervening value in the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of the included limits are also encompassed within the present invention.

[0048] Certain ranges of values ​​presented herein are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that follows it, as well as a number that is close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context presented, provides a substantial equivalent to the specifically recited number.

[0049] 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. Representative illustrative methods and materials are now described, 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.

[0050] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the publication cited. 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, publication dates provided may be different from actual publication dates, which may need to be independently confirmed.

[0051] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for using exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for using a "negative" limitation.

[0052] As will be apparent to those skilled in the art after reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any one of the other several embodiments without departing from the scope or spirit of the invention.

[0053] Any recited method may be performed in the order of events recited or in any other order that is logically possible. The apparatus and method have been or will be described for grammatical fluency and functional explanation, but it should be expressly understood that the claims should not be construed as necessarily limited in any way to the interpretation of the "means" or "step" definition unless expressly recited under 35 U.S.C. §112, but should be given the full meaning and scope of equivalents provided by the definitions in the claims under the doctrine of equivalents, and the claims that expressly recite 35 U.S.C. §112 should be given all legal equivalents under 35 U.S.C. §112.

[0054] Systems for processing cell samples

[0055] As described above, aspects of the present invention include systems for processing cell samples. In some embodiments, the system includes structures or features intended to facilitate the execution of a workflow. In some embodiments, the workflow may include a series of steps for hybridizing nucleic acids from multiple cells with nucleic acids (e.g., barcoded nucleic acids) displayed on the surfaces of multiple barcoded beads. For example, in some embodiments, the workflow may include the following steps: introducing multiple cells into a microwell array; introducing multiple beads carrying barcoded nucleic acids into a microwell array; lysing multiple cells so that nucleic acids from the cells (e.g., mRNA from the cells) are hybridized with barcoded nucleic acids associated with (e.g., present on the beads surface) multiple beads; and then collecting beads from the microwell array. In some cases, this workflow is part of a next-generation sequencing (NGS) library preparation workflow.

[0056] As described above, the system of the present invention includes a tray configured to receive a multi-microporous array flow cell box, and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the box when in the enabled position. "Uniform magnetic force" means that the magnetic force is uniformly applied to the application area of ​​the multi-microporous array flow cell box. In other words, the magnetic force applied to a certain part of the multi-microporous array flow cell box is very small compared to the magnetic force applied to another part of the multi-microporous array flow cell box, for example, a difference of 20% or less, for example, a difference of 15% or less, for example, a difference of 10% or less, for example, a difference of 5% or less, and including a difference of 1% or less. In an embodiment, the uniform magnetic force of the present invention causes the magnetic force applied to each flow cell of the box to be comparable (including the same) when the multi-microporous array flow cell box is placed in the system, for example, the magnetic force applied to any two given flow cells differs by no more than 20%, for example, no more than 15%, for example, no more than 10%, for example, no more than 5%, and including a difference of no more than 1%. The strength of the uniform magnetic force can vary. For example, in some cases, the uniform magnetic force is a magnetic field in the range of 650 Gauss to 1,325 Gauss, for example, 650 Gauss to 700 Gauss, for example, 700 Gauss to 750 Gauss, for example, 750 Gauss to 800 Gauss, for example, 800 Gauss to 850 Gauss, for example, 850 Gauss to 900 Gauss, for example, 900 Gauss to 950 Gauss, for example, 950 Gauss to 1,000 Gauss, for example, 1,000 Gauss to 1,050 Gauss, for example, 1,050 Gauss to 1,200 Gauss, for example, 1,200 Gauss to 1,250 Gauss, for example, 1,250 Gauss to 1,300 Gauss, and including 1,300 Gauss to 1,325 Gauss.

[0057] In an embodiment, the tray is located on the upper surface of the body of the subject system. In some embodiments, the tray can receive a multi-micropore array flow cell box within the system. In some embodiments, the size, position, or other configuration of the tray can orient the multi-micropore array flow cell box to a predetermined position within the system. The predetermined position can facilitate the interaction between other components of the loading station and the multi-micropore array flow cell box. In some embodiments, the tray can releasably fix the box within the system. For example, in some embodiments, the shape of the tray complements the shape of the multi-micropore array flow cell box so that the multi-micropore array flow cell box can be received in the tray in a single orientation. For example, in some cases, if the multi-micropore array flow cell box includes one or more chamfers (described in more detail below), the tray has a corresponding shape, that is, it is configured to receive a box with a chamfer. In this way, the chamfer can be used as a directional feature for box installation.

[0058] In some cases, the system includes one or more retaining mechanisms that are configured to maintain the box in place when the multi-micropore array flow cell box is placed in a tray. In some such cases, the tray includes a latch for holding the multi-micropore array flow cell box. In selected embodiments, the latch is spring-loaded. When the box is inserted, the spring-loaded latch can be pushed back and force is applied to the box to limit the box in place. In some cases, the latch includes one or more protruding features that allow the user to slide the latch backward when removing the box. The latch can be constructed using any convenient technology. In some embodiments, the latch is injection molded. In some such embodiments, the latch includes an injection moldable polymer. Any convenient injection moldable polymer can be used. Injection moldable polymers 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), polyphenylene sulfone (PPSU), polyetheretherketone (PEEK), and polyetherimide (PEI). If the latch is spring-loaded, in some cases, the spring may be made of stainless steel (e.g., 18-8 stainless steel) to prevent oxidation due to nearby liquids. In some cases, the latch additionally includes a latch plate configured to anchor the latch to the system. For example, in some cases, the latch plate is configured to anchor the latch to the system via one or more screws. If a latch plate is present, the latch plate can be made of any suitable material. In some cases, the latch plate is made of metal 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 some embodiments, the latch plate is made of aluminum, such as 6061 aluminum.

[0059] In an embodiment, the tray also includes a box lock catch. The box lock catch can be positioned on the opposite side of the tray relative to the latch, and provides a downward force to the box when the box is placed in the tray. The downward force can be enough to prevent the box from being removed from the tray until the latch is opened. The box lock catch can be anchored to the system in any convenient manner, for example, by one or more screws. If present, the box lock catch can be made of any suitable material. In some cases, the box lock catch is made of metal material, including but not limited to aluminum, titanium, brass, iron, lead, nickel, steel (for example stainless steel), copper, tin and combinations and alloys thereof. In an embodiment, the box lock catch is made of aluminum, such as 6061 aluminum.

[0060] The system of interest also includes a retrieval magnet assembly. As described above, the retrieval magnet assembly is configured to apply a uniform magnetic force to the flow cell of the box when in the enabled position. For example, when it is necessary to retrieve the beads in the micropores of the box, the retrieval magnet assembly can be used. The retrieval magnet assembly can be switched between the enabled position and the disabled position. The enabled position refers to the position where the retrieval magnet assembly applies a magnetic force to the flow cell of the box, and the disabled position refers to the position where the retrieval magnet assembly applies a smaller magnetic force (including no magnetic force) to the flow cell of the box compared to the enabled position. In the disabled position, compared to the enabled position, the retrieval magnet assembly is farther away from the multi-micropore array flow cell box in at least one direction. In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force to the multi-micropore array flow cell box from a position above the tray. In some embodiments, when in the enabled position, the lower surface of the retrieval magnet assembly can be parallel to the upper surface of the multi-micropore array flow cell box. In selected cases, when in the enabled position, the lower surface of the retrieval magnet assembly can be 1.0 mm or about 1.0 mm away from the upper surface of the multi-micropore array flow cell box. In selected cases, the distance between the lower surface of the retrieval magnet assembly and the upper surface of the multi-micropore array flow cell box can 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, not more than 1.0 mm, 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. In some embodiments, the system can include a retrieval magnet assembly driver. In some embodiments, the driver can be driven to convert the retrieval magnet assembly between the inactive position and the enabled position.

[0061] Although two positions of the retrieval magnet assembly are described above, it should be appreciated that in certain embodiments, the retrieval magnet assembly can be moved between more than two positions. In some embodiments, the retrieval magnet assembly can be moved between discontinuous positions. In some embodiments, the retrieval magnet assembly can be moved within a continuous range of positions. In an embodiment, when the multi-microwell array flow cell box is located in a tray, the position of the retrieval magnet assembly can allow magnetic forces of different sizes to be applied to the magnetic particles in the box.

[0062] In some embodiments, the system does not include a lysis magnet in a position below the tray. A "lysis magnet" refers to a magnet that is effective in the lysis step of a cell sample preparation method. For example, when the lysis buffer is pumped through the flow cell, the lysis magnet can pull the beads into and hold them in the micropores. For example, the magnet 120 disclosed in U.S. Patent Nos. 10,634,691 and 11,061,043 is considered to be a lysis magnet herein, which is not present in the embodiments of the subject system. Before the present disclosure was submitted, it was recognized that a lysis magnet was needed to prevent the beads from being washed away. However, the inventors unexpectedly realized that the lysis magnet could be removed from the design and that the beads would remain in the micropores during the application of the lysis buffer.

[0063] In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force via a plurality of magnets. In such a case, the number of magnets in the plurality of magnets can vary and can range from 2 to 10, such as 2 to 6, and including 3 to 5. For example, the number of magnets can be 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some cases, the retrieval magnet assembly comprises 4 magnets. The magnets in the retrieval magnet assembly can comprise any suitable magnets. Magnets of interest include, but are not limited to, rare earth magnets such as neodymium magnets, samarium cobalt magnets, and combinations thereof. The size and shape of the magnets can also vary. In some cases, the magnets are bar magnets. When the magnets are bar magnets, the length of each bar magnet can vary, and in some cases, its length can range from 50 mm to 500 mm, such as 60 mm to 200 mm, such as 70 mm to 80 mm, and for example, including 75 mm to 125 mm. In some cases, the length of the bar magnet is 76.2 mm. The width of the bar magnet can also vary, and in some cases, its length can range from 5mm to 20mm, such as 6mm to 18mm, including, for example, 10mm to 15mm. In some cases, the width of the magnet is 12.7mm. The thickness of the magnet can also vary, and in some cases, its length ranges from 0.5mm to 10mm, such as 1mm to 5mm, including, for example, 2mm to 4mm. In some cases, the width of the magnet is 3.175mm. In other embodiments, the magnet is an annular magnet. In some such embodiments, the annular magnets are concentrically arranged, such as in a bull's-eye configuration. The magnets in the plurality of magnets can have the same shape or different shapes. In some embodiments, the magnets have different shapes and / or sizes.

[0064] The relative positions of the magnets in the retrieval magnet assembly can vary. In some cases, the distance between adjacent magnets in a plurality of magnets ranges from 0.1 mm to 20 mm, for example, from 1 mm to 15 mm, and including 1 to 9 mm. In some cases, adjacent magnets can be spaced 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 distances between adjacent magnets can be the same or different. In some cases, each magnet in the retrieval magnet assembly is spaced the same distance from the adjacent magnets. In other cases, the distance between at least one magnet and the adjacent magnets is different from the distance between another pair of adjacent magnets. In an example involving four bar magnets, the distance between the first and second magnets is 2 mm, the distance between the second and third magnets is 8 mm, and the distance between the third and fourth magnets is 2 mm.

[0065] In an embodiment, the plurality of magnets have alternating polarity. Without being bound by theory, it is believed that alternating the polarity of the magnets allows the magnetic field lines from the north pole of an adjacent magnet to pass through the box before entering the south pole. For example, in some embodiments, adjacent magnets in the retrieval magnet assembly are arranged in an N, S, N, S ... configuration, where "N" represents the north pole and "S" represents the south pole, and this pattern can be repeated for all magnets in the plurality of magnets. In an alternative embodiment, the magnets in the retrieval magnet assembly are arranged in an S, N, S, N ... configuration.

[0066] Figures 1A-1E Depicted are a retrieval magnet assembly and tray according to certain embodiments of the present invention. Figure 1A and Figure 1B Different views of the system 100 are shown, wherein the retrieval magnet assembly 101 is in an inactive position. The retrieval magnet assembly 101 is located at a distance from the tray 102. Figure 1C and Figure 1D Different views of the system 100 are shown with the retrieval magnet assembly 101 in an activated position, wherein the retrieval magnet assembly 101 is located adjacent to the tray 102 . Figure 1E Another view of the system 100 is shown showing a latch 104, a multi-microwell array flow cell cartridge 106, a latch plate 105, and a cartridge latch 103. The latch 104 and cartridge latch 103 are configured to retain the multi-microwell array flow cell cartridge 106 within the tray 102. The latch 104 is spring-loaded and can be pushed back to remove the multi-microwell array flow cell cartridge 106.

[0067] Figures 2A-2B Different views of a retrieval magnet assembly are shown, according to certain embodiments. Figure 2AThe retrieval magnet assembly 201 is depicted in an activated position. The retrieval magnet assembly 201 is located in an upper position on the tray 202. Figure 2B Depicted are multiple magnets within retrieval magnet assembly 201. Magnets 203a-203d are bar magnets with alternating polarity and are arranged in an N, S, N, S configuration.

[0068] In some embodiments, the system further comprises a sample collection container support, which is configured to receive a plurality of sample collection containers for collecting analytes from the box. The sample collection container support is configured to be received in the system of the present invention at a position lower than a tray (and box (if present)), and positions the sample collection container so that the liquid from the box can be received in the sample collection container. In selected embodiments, the sample collection container support comprises a counterweight, which is configured to maintain the sample collection container support in an upright position. In other words, when the sample collection container support is removed from the system, the sample collection container support is not easy to overturn. In some cases, the counterweight can be positioned at a position lower than that receiving the sample collection container. In some cases, the system (for example, the drawer of the system) and the sample collection container support have complementary shapes so that the sample collection container support can be received in the system with a single orientation. In other words, the sample collection container support has a mistake-proofing design. More details about the sample collection container support of the present invention will be provided below.

[0069] In some embodiments, the system is further included in a plurality of sample collection containers within a sample collection container holder. The sample collection container can be any suitable liquid container. Suitable sample collection containers include, but are not limited to, test tubes, conical tubes, multi-compartment containers (e.g., microtiter plates (e.g., 96-well plates)), centrifuge tubes, culture tubes, microtubes, bottle caps, cuvettes, bottles, linear polymer containers and bags, and other types of containers. In some cases, the sample collection container is a test tube. In some cases, the opening of each collection container can include a lid (e.g., a valve), which can be reversibly closed as needed. The sample collection container holder can accommodate any suitable number of sample collection containers, e.g., the number of sample collection containers ranges from 2 to 10, including 7 to 9. In some cases, the system comprises 8 sample collection containers.

[0070] In some cases, the system further comprises a waste collection container for collecting liquid waste from the multi-microwell array flow cell cartridge. The waste collection container can include, but is not limited to, conical tubes, centrifuge tubes, culture tubes, bottles, linear polymer containers and bags, and other types of containers.

[0071] In some embodiments, system of the present invention comprises a drawer, and this drawer is configured to hold one or more articles, and this drawer can be moved between a plurality of different positions.In some cases, this drawer is configured to receive at least one in sample collection container support and waste collection container.In some such cases, this drawer is configured to receive both sample collection container support and waste collection container.In some embodiments, this drawer can be moved to at least one position, and in this position, at least one (including all) sample collection container and / or waste collection container in the sample collection container support is aligned with one or more outlets of box.In some embodiments, this system comprises a drawer driver, and this drawer driver can be moved between a plurality of different positions, and wherein at least some movement of drawer driver can cause the movement of drawer.In some embodiments, drawer can be moved along guide rail.In some embodiments, guide rail is positioned to engage with the top surface, side or bottom surface of drawer.In some embodiments, guide rail is positioned to reduce the contact of overflowing or splashing liquid.

[0072] The system may also include a drip tray designed to cover the linear rails and carry liquid when the box is not above a waste or sample collection container. In some cases, the drip tray is detachable and removable (e.g., for cleaning). In selected cases, the drip tray has higher edge walls and an ergonomic handle for pipetting to ensure that the liquid remains in the tray. In some cases, the drip tray is movable along the guide rails and may be driven by a drawer drive.

[0073] Figures 3A-3H Different views of a system for processing a cell sample according to certain embodiments of the present invention are shown. Figure 3A A front view of the system 300 is shown, Figure 3B A rear view of the system 300 is shown. Figure 3C A top view of the system 300 is shown, Figure 3D A bottom view of the system 300 is shown, Figures 3E-3F A 45° angle view of the system 300 is shown, and Figures 3G-3HA side profile view of system 300 is shown. System 300 includes a retrieval magnet assembly 301, a tray 302, a retrieval magnet assembly driver 303, a latch 304, a sample collection container holder 305, a waste collection container 306, a drawer driver 307, a drawer 308, and a drip tray 309. The retrieval magnet assembly 301 can be driven to an enabled position by the retrieval magnet assembly driver 303. When in the enabled position, the retrieval magnet assembly 301 is configured to apply a uniform magnetic force to the multi-micropore array flow cell box located in the tray 302. System 300 also includes a latch 304, which is configured to maintain the multi-micropore array flow cell box in the tray 302. In this embodiment, the latch 304 is a spring-loaded latch that is configured to push back when the multi-micropore array flow cell box is loaded into the tray 302 and apply force to the box to maintain its position.

[0074] exist Figures 3A-3H In the example of FIG, the drawer 308 includes a sample collection container holder 305 and a waste collection container 306. The drawer 308 has grooves that are complementary in shape to the sample collection container holder 305 and the waste collection container 306, so that the sample collection container holder 305 and the waste collection container 306 can only be fitted into the drawer 308 in a single orientation. The drawer 308 is operably connected to a drawer drive 307 that is configured to move the drawer 308 between different positions within the system 300. For example, the drawer 308 can be moved to a first position in which the drawer is located outside the system 300 (e.g., Figures 3A-3H ). In the first position, waste collection containers, sample collection containers, and / or sample collection container holders can be added, removed, or replaced as needed. Using the drawer drive 307, the drawer 308 can be moved to a sample collection position, in which liquid can be received in one or more sample collection containers within the sample collection container holder 305; or to a waste retrieval position, in which liquid can be received in the waste collection container 306. The drawer 308 can also be moved to one or more intermediate positions via the drawer drive 307, in which the drawer is positioned within the system 300 but is not used to receive liquid. Figure 3F Depicted is a drip tray 309 configured to collect liquid not received in a sample collection container or a waste collection container.

[0075] Figures 4A-4B Depicted are the drip tray and the manner in which the drip tray and drawer are moved through in the system of the present invention. Figure 4A A drip tray 400 is shown that includes an ergonomic handle 402 to facilitate removal and / or manipulation of the drip tray 400 and a high boundary wall 401 to ensure that liquid remains within the drip tray. Figure 4BThe position of the drip tray 400 relative to the other components of the system is depicted according to certain embodiments. Figure 4B As shown, the tray 400 is operably connected to a drawer drive 407 such that movement of the drawer drive 407 moves the drawer 408 (along with the sample collection container holder 405 and the waste collection container 406 (if present)) and the tray 400 along the guide rails 410 through the system.

[0076] Aspects of this system additionally include an interlocking device, which is configured to prevent sample liquid from being collected in a waste collection container when the retrieval magnet assembly is in the enabled position. As described above, when it is necessary to obtain sample liquid from the box (for example, in one or more sample collection containers), the retrieval magnet assembly can be driven to the enabled position (for example, by user operation). The inventors have found that a common mistake made by users of conventional systems is that sometimes samples are retrieved into a waste container instead of a sample collection tube. The interlocking device described herein is configured to prevent erroneous retrieval by locking the drawer in place during the retrieval operation. In other words, when the retrieval magnet is in the enabled position, the drawer cannot move because the interlocking device limits its movement. In some embodiments, the drawer is limited to a position where the sample collection container (if present) in the sample collection container holder is located below the box outlet so that the sample liquid is received in the sample collection container. In some cases, the retrieval magnet assembly is operably connected to the interlocking device so that the movement (i.e., driving) of the retrieval magnet assembly also causes the interlocking device to move. The interlock engages the drawer and prevents it from moving (e.g., moving along the rails). Moving the retrieval magnet assembly back to the inactive position causes the interlock to disengage the drawer, allowing the drawer to move freely when driven by the drawer driver.

[0077] Figures 5A-5B An interlock according to certain embodiments is depicted.System 500 includes a retrieval magnet assembly 501 , a retrieval magnet assembly driver 503 , a drawer driver 507 , a drawer 508 , and an interlock 511 operably connected to the retrieval magnet assembly 501 . Figure 5A The retrieval magnet assembly 501 is shown in the deactivated position. The interlock 511 is not engaged with the drawer 508 and the drawer is free to move via the drawer drive 507. Figure 5B The retrieval magnet assembly 501 is shown in an activated position. The interlock 511 is engaged with the drawer 508 so that movement of the drawer 508 is restricted.

[0078] Multiwell Array Flow Cell Kit

[0079] As described above, aspects of the present invention also include multi-micropore array flow cell cassettes. The multi-micropore array flow cell cassettes described herein (also referred to herein as "cassettes") comprise multiple fluid lanes, each of which comprises an inlet for receiving liquid, a flow cell comprising a micropore array, and an outlet for discharging the liquid. "Multi-micropore array flow cell" means that the cassette has multiple (i.e., many) flow cells, and each flow cell comprises a micropore array. Each flow cell is a component of a fluid lane (i.e., the path for fluid to flow from the inlet to the outlet). The micropore array of the subject cassette, when used for cell capture, can achieve a cell capture rate of 50% or higher, such as 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, and including 95% or higher. In some cases, the cassettes of the present invention can be reused. For example, if one lane of the cassette is used for a specific assay, the remaining lanes of the cassette can be used for the same or different assays at one or more different time points. In some cases, a single multi-microwell array flow cell cartridge can be used 1 time or more, such as 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, and including 8 times or more. In selected cases, a partially used cartridge (i.e., a cartridge in which one or more channels have been used for determination) can be stable (i.e., remain operable) for 1 month or longer, such as 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, and including 6 months or longer. In some embodiments, the use of a multi-microwell array flow cell cartridge can minimize batch effects between fluid lanes, or even eliminate batch effects altogether.

[0080] The number of fluid lanes of the box can vary. In some cases, the multi-micropore array flow cell box comprises a number ranging from 2 to 20 fluid lanes, such as 2 to 15, and including 2 to 10. For example, in an embodiment, the box comprises 2 lanes, 3 lanes, 4 lanes, 5 lanes, 6 lanes, 7 lanes, 8 lanes, 9 lanes or 10 lanes. In certain embodiments, the multi-micropore array flow cell box comprises 8 fluid lanes. Each fluid lane comprises a flow cell, which comprises a micropore array. Micropore array and flow cell will be described in detail below. In some cases, the box comprises multiple flow cell subsets. In other words, multiple fluid lanes can comprise smaller fluid lane sets, which can be manufactured separately. Each subset can comprise 2 to 6 flow cells, such as 3 to 5. In some cases, each subset comprises 4 flow cells.

[0081] The entrance of each fluid swimming lane can change. In some cases, the entrance comprises a gasket. In some cases, the entrance comprises a gasket, which is configured to form a tapered lock (for example, when the pipette tip is inserted into the entrance, liquid can be introduced into the fluid swimming lane) on the side of the pipette tip. The configuration of the gasket can make the overall Z-axis tolerance range of the tapered lock be 0.1mm to 1mm, for example 0.2mm to 0.8mm, for example 0.3mm to 0.7mm, and include 0.4mm to 0.6mm. In some cases, the overall Z-axis tolerance of the tapered lock is 0.5mm. In the case of selection, the Z-axis tolerance is enough to cope with the height difference between different pipette tips. The hardness of the gasket can change. In some cases, the Shore hardness range of the gasket is 1 to 100, for example 5 to 90, and include 10 to 80. The inlet can be constructed of any suitable material, including but not limited to silicon, fused silica, glass, various 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 polytetrafluoroethylene (PTFE), metals (such as aluminum, stainless steel, copper, nickel, chromium and titanium), or any combination thereof.

[0082] The outlet of each fluid lane can also change similarly. In some cases, the configuration of the outlet can induce the formation of droplets. In some such cases, the configuration of the outlet can induce droplet formation within the range of fluid flow rate of 5 μL / s to 1,000 μL / s, for example, 10 μL / s to 700 μL / s, 15 μL / s to 600 μL / s, and including 20 μL / s to 500 μL / s. In a selected embodiment, the outlet is a tapered orifice. The orifice is designed to adapt to various fluids with different surface energies by specific inner diameter, outer diameter, cone angle and length, and maintain uniform fluid droplets. In some cases, the inner diameter range of the outlet is 0.500mm to 1.200mm, for example 0.550mm to 0.650mm, and including 0.750mm to 1.050mm. In some cases, the outer diameter of the outlet is 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 cases, the cone angle of the outlet is 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 length of the outlet is 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 can be constructed of any suitable material, including but not limited to silicon, fused quartz, glass, various 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 polytetrafluoroethylene (PTFE), metals (such as aluminum, stainless steel, copper, nickel, chromium and titanium), or any combination thereof.

[0083] In some cases, the outlet is a stepped outlet. "Stepped" means that the outlet includes a raised portion through which fluid must pass before exiting. In selected embodiments, the stepped shape of the outlet is sufficient to prevent siphoning of liquid from the flow cell. In some cases, the outlet is provided with a siphon cap. In this case, the siphon cap is configured to form a channel along the raised portion of the stepped outlet to guide fluid to the outlet for exit.

[0084] The inlet and outlet features of the box can be designed to provide a convenient and leak-proof fluid connection with the instrument, or can be used as an open reservoir for manually moving samples and reagents in or out of the box. Examples of convenient mechanical design of inlet and outlet port connectors include, but are not limited to, threaded connectors, Luer lock connectors, Luer slip or "sliding tip" connectors, press-fit connectors, etc. The inlet and outlet of the box can also include a cap, a spring-loaded lid or closure, or a polymer film that can be opened or punctured when the box is placed in the instrument, and is used to prevent the box's internal surface from being contaminated during storage, or to prevent fluid from overflowing when the box is taken out from the instrument. The outlet of the box can also include a removable sample collection chamber that is suitable for interacting with a stand-alone PCR thermal cycler or a sequencer.

[0085] In some embodiments, the inlet and outlet can be capable of directing fluid through the fluid channel, thereby contacting the fluid with the micropore. In some embodiments, the device includes a pipette tip interface for loading or removing a cell sample, assay reagent, bead suspension, waste liquid, or a combination thereof from the device. The device can contain a cell sample, an assay reagent, a bead suspension, or a combination thereof.

[0086] The box of the present invention can additionally comprise an outer shell. The outer shell of interest is configured to accommodate the components of the box, such as a flow cell, an inlet and an outlet. The outer shell can be made of any suitable material, including but not limited to silicon, fused quartz, glass, various polymers such as polydimethylsiloxane (PDMS; elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), polyimide, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, non-stick materials such as polytetrafluoroethylene (PTFE), metals (such as aluminum, stainless steel, copper, nickel, chromium and titanium) or any combination thereof. In some cases, the outlet is contained in the outer shell.

[0087] Figures 6A-6C An embodiment of the presently claimed cartridge is depicted. Figure 6A An exploded view of a cartridge is shown, comprising a plurality of flow cells 601, a gasket 602, and a housing 603. Also shown is a siphon cap 604, which is configured to be placed adjacent an outlet within the housing 603 to form a channel over a raised portion of the outlet. Figure 6B Another view of the gasket 602 is shown. Figure 6B As shown, the gasket 602 forms a conical lock around the pipette tip 605 when liquid is loaded into the fluid lanes of the box. Figure 6C An outlet 607 is shown having a tapered orifice.

[0088] Figures 7A-7HShown are different views of a multi-microwell array flow cell cartridge according to certain embodiments. Figures 7A-7B A side profile view of a multi-well array flow cell cartridge 700 is shown. Figure 7C 7 is a top view of cartridge 700. Cartridge 700 shows inlets 701a-701h, flow cells 702a-702h, and outlets 703a-703h. Figure 7D 700 is a bottom view of the box.

[0089] Figure 7E A 45° angle view of the top of the box 700 is shown, and Figure 7F A 45° angle view of the bottom of the box 700 is shown. Figure 7G and Figure 7H A side profile view of the cartridge 700 is shown.

[0090] In some cases, the box includes one or more alignment features that are configured to facilitate the assembly of the box and the positioning of the box in the tray of the present invention system. For example, in some embodiments, the box of the present invention includes a chamfer (i.e., a transition edge between two edges of the box). In some cases, the chamfer may be characterized by a certain angle relative to the straight edge of the box. In some such cases, the angle range is 30° to 60°, such as 35° to 55°, such as 40° to 50°, and includes 44° to 46°. As described above, the tray can be configured so that the box can only be placed in the tray in a single direction. In other words, the tray and the box have corresponding shapes. The box can additionally include an aligner so that the flow cell can only be attached to the housing in a single direction. In some cases, the aligner can take the form of a protrusion in the box housing. In some embodiments, the tray and the box comply with the ANSI / SLAS microplate standards. These standards can be consulted at https: / / www(dot)slas(dot)org / education / ansi-slas-microplate-standards / .

[0091] Figure 8 A bottom view of a multi-microwell array flow cell cartridge according to certain embodiments of the present invention is shown, along with alignment features associated therewith. The cartridge 800 includes a chamfer 801 configured to orient the cartridge 800 in a tray (not shown) of the system of the present invention. The cartridge 800 also includes aligners 802 and 803 configured to align a flow cell assembly 804 when installed.

[0092] flow cell

[0093] The design of each flow pool can include multiple microarray chambers that interface with multiple microwell arrays so that one or more different cell samples can be processed in parallel. The design of the flow pool can also include features for generating a consistent (e.g., uniform) flow velocity profile (i.e., "plug flow") across the entire width of the array chamber so that cells and beads are delivered to the microwells more efficiently (e.g., uniformly), for example, by using a porous barrier located near the chamber inlet and upstream of the microwell array as a "flow diffuser," or by dividing each array chamber into several sub-regions that collectively cover the same total array area, but separate inlet fluid streams flow through these sub-regions in parallel. In some embodiments, the flow pool can encapsulate or integrate more than one microwell array substrate.

[0094] Typically, the size of the fluid channel and array chamber in the flow cell design will be optimized to (i) efficiently (e.g., evenly) deliver cells and beads to the microwell array, and (ii) minimize the consumption of sample and reagent. In an embodiment, each flow cell comprises an elongated channel. In some cases, the length of the elongated channel can be characterized by a straight line, i.e., the channel does not have a curve. The length of the elongated channel can vary. In some cases, the length range is 20mm to 500mm, e.g., 25mm to 400mm, e.g., 30mm to 300mm, e.g., 40mm to 300mm, e.g., 45mm to 200mm, and includes 50mm to 100mm. The width of the elongated channel can be different in different embodiments, e.g., ranging from 0.1mm to 100mm. In some embodiments, the width can be or be about 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 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.

[0095] The height of the fluid channel can be different in different embodiments, for example, ranging from 0.1 mm to 100 mm. In some embodiments, the height can be or 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, 3, 4, 5, 6, 7, 8, 9, 10 mm, or a number or range between any two of 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.

[0096] The flow cell can be manufactured using a variety of techniques and materials known to those skilled in the art. Typically, the flow cell can be manufactured as a separate component and then mechanically clamped or permanently bonded to the microwell array substrate. Examples of suitable manufacturing techniques include conventional machining, CNC machining, injection molding, 3D printing, alignment and lamination of one or more layers of laser or die-cut polymer films, or any of a variety of microfabrication techniques, such as photolithography and wet chemical etching, dry etching, deep reactive ion etching, or laser micromachining.

[0097] Once the flow cell components are fabricated, they can be mechanically attached to the microwell array substrate, for example by clamping them to the microwell array substrate (with or without gaskets), or they can be directly bonded to the microwell array substrate using various techniques known to those skilled in the art (depending on the choice of materials used), for example by using anodic bonding, thermal bonding or various adhesives or adhesive films, including epoxy, acrylic, silicone, UV-curable, polyurethane or cyanoacrylate adhesives. In some embodiments, the substrate can form the bottom of the fluid channel, or the substrate can be located at the bottom of the fluid channel. In some embodiments, the substrate comprises silicon, fused quartz, glass, polymer, metal, elastomer, polydimethylsiloxane, agarose, 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 selected cases, the flow cell is made by bonding the microwell substrate to the fluid substrate.

[0098] Flow cell can be manufactured using various materials well known by persons skilled in the art. Usually, the selection of materials used will depend on the manufacturing technology adopted, and vice versa. Suitable materials include but are not limited to silicon, fused silica, glass, various polymers such as polydimethylsiloxane (PDMS; Elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), polyimide, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, metal (such as aluminum, stainless steel, copper, nickel, chromium and titanium), non-stick materials such as polytetrafluoroethylene (PTFE) or the combination of these materials. Cyclic olefin polymer (COP) can comprise Zeonor 1020R or Zeonor 1060R.

[0099] Microwell array

[0100] In some embodiments, the micropore can include a small reaction chamber with a limited volume. In some embodiments, the micropore can capture one or more cells. In some embodiments, the micropore can only capture one cell. In some embodiments, the micropore can capture one or more solid supports (e.g., beads). In some embodiments, the micropore can only capture one solid support. In some embodiments, the micropore captures a single cell and a single solid support (e.g., beads). In some embodiments, the micropore can accommodate a single particle (e.g., a cell or a bead). In some embodiments, the micropore can accommodate two different particles (e.g., a cell and a bead).

[0101] Micropore can be made into various shapes. Non-limiting exemplary hole geometry can include cylindrical, conical, hemispherical, rectangular or polyhedral (for example, the three-dimensional geometry of being made up of multiple planes, for example, hexagonal column, octagonal column, inverted triangular pyramid, inverted quadrangular pyramid, inverted pentagonal pyramid, inverted hexagonal pyramid or inverted truncated cone). Micropore can comprise the shape of combining two or more of these geometric shapes. For example, micropore can be partially cylindrical, and the remainder is inverted cone. Micropore can comprise two cylinders side by side, wherein the diameter of one cylinder is larger (for example, roughly equivalent to the diameter of bead), and the other is smaller (for example, roughly equivalent to the diameter of cell), and they are connected by vertical channel (i.e., parallel to the cylinder axis), and this channel extends to the whole length (depth) of cylinder. The position of micropore opening can change. For example, the opening of micropore can be located at the upper surface of substrate. For example, the opening of micropore can be located at the lower surface of substrate. The shape of micropore closed end (for example, bottom) can change. For example, the closed end of micropore can be flat. For example, the closed end of the micropore can have a curved surface (e.g., convex or concave). The shape and / or size of the micropore can be determined based on the type of cells or solid support to be captured. In some embodiments, the cross-section of the micropore in the plane of the substrate can be non-circular (e.g., square or hexagonal).

[0102] Micropore can be made into a variety of sizes. Micropore size can be characterized by the diameter and / or depth of, for example, micropore. The diameter of micropore can refer to the largest circle that can be inscribed in the plane cross section of micropore geometry. In some embodiments, the diameter range of micropore can be about 1 times to about 10 times of the cell to be captured in micropore or the diameter of solid support. In some embodiments, micropore diameter can be or be about 1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times of the cell to be captured in micropore or the diameter of solid support, or the numerical value or scope between any two of these values. In some embodiments, micropore diameter can be at least or at most 1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times of the cell to be captured in micropore or the diameter of solid support. In some embodiments, micropore diameter can be about 2.5 times of the cell to be captured in micropore or the diameter of solid support.

[0103] Micropore diameter can be represented by absolute size.The scope of micropore diameter can be about 1 nanometer to about 1000 micron.In some embodiments, micropore diameter can be or be about 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 micron, or the numerical value or scope between any two of these values.In some embodiments, micropore diameter can be at least or at most 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900,1000 micron. In some embodiments, the micropore diameter can be or be about 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 microns, or a numerical value or range between any two of these values. In some embodiments, the micropore diameter 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, 1,000 microns. In some embodiments, the micropore diameter can be about 30 microns.

[0104] In some embodiments, the depth of micropores can be less than the diameter of beads. For example, the depth of micropores can be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%, 100% of the bead diameter, or a numerical value or range between any two of these values. For example, the depth of micropores can be at least or at most 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%, 100% of the bead diameter. In some embodiments, synthetic particles (eg, beads) may protrude outside of the microwells.

[0105] In some embodiments, the size of the micropore allows the micropore to accommodate at most one bead. The ratio of micropore width to bead diameter can vary in the range of 1-1.9. In some embodiments, the ratio of micropore width to bead diameter can be or be about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or a numerical value or range between any two of these values. In some embodiments, the ratio of micropore width to bead diameter can 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.

[0106] In some embodiments, the micropore depth can be or be approximately 1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times of the cell to be captured in the micropore or the solid support diameter, or a numerical value or scope between any two of these values. In some embodiments, the micropore depth can be approximately 2.5 times of the cell to be captured in the micropore or the solid support diameter.

[0107] The aspect ratio of the micropore width to the micropore depth can vary, for example, in the range of 0.1-2. In some embodiments, the aspect ratio of the micropore width to the micropore depth can be or is 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 value or range between any two of these values. In some embodiments, the aspect ratio of the micropore width to the micropore depth 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, or 2.

[0108] In some embodiments, the depth of micropore can be represented by its absolute size.For example, the depth range of micropore can be from about 1 nanometer to about 1000 micron.In some embodiments, the micropore depth can be or be about 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 micron, or the numeral or scope between any two of these values.In some embodiments, the micropore depth can be at least or at most 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900,1,000 micron. In some embodiments, the micropore depth can be or is about 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 microns, or a number or range between any two of these values. In some embodiments, the micropore depth 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, 1,000 microns. In some embodiments, the micropore depth ranges from 20 microns to 60 microns, such as 25 microns to 55 microns, such as 30 microns to 50 microns, and including 45 microns to 49 microns. In some cases, the micropore depth is 48 microns.

[0109] The volume of the micropores can vary, for example, in the range of about 1 picoliter to about 1,000 microliters. In some embodiments, the micropore volume can be or is about 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 picoliters, or a value or range between any two of these values. In some embodiments, the micropore volume 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 picoliters. In some embodiments, the micropore volume can be or is about 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 nanoliters, or a number or range between any two of these values. In some embodiments, the micropore volume 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 nanoliters. In some embodiments, the micropore volume can be or be about 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 microliters, or a number or range between any two of these values. In some embodiments, the micropore volume 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 microliters. In some embodiments, the micropore volume can be about 1 microliter.

[0110] Micropore volume can be characterized by the volume difference between different micropores.The coefficient of variation of micropore volume (expressed as a percentage) can be from about 1% to about 100%.The coefficient of variation of micropore volume can be or be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or the numerical value or scope between any two of these values.The coefficient of variation of micropore volume 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 micropore volume can be about 2.5%.

[0111] The ratio of microwell volume to bead surface area (or to the surface area of ​​the solid support to which random barcode oligonucleotides can be attached) can vary, for example, in the range of about 2.5 to about 1,520 microns. In some embodiments, the ratio can be or be about 2.5, 5, 10, 100, 500, 750, 1000, 1,520 microns, or a value or range between 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 microns. In some embodiments, the ratio can be about 67.5 microns.

[0112] The microwells can be arranged in one-dimensional, two-dimensional or three-dimensional arrays. A three-dimensional array can be achieved, for example, by stacking a series of two or more two-dimensional arrays (eg, stacking two or more substrates containing microwell arrays).

[0113] The pattern and spacing between the microwells can be varied to optimize the efficiency of capturing single cells and single solid supports (e.g., beads) in each well, as well as to maximize the number of wells per unit area of ​​the array. The microwells can be distributed in a variety of random or non-random patterns. For example, they can be completely randomly distributed on the surface of the array substrate, or can be arranged in a square grid, a rectangular grid, a hexagonal grid, etc.

[0114] The center-to-center distance or center-to-center spacing between the holes can vary from about 1 micron to about 1,000 microns. In some embodiments, the center-to-center distance between the holes can be or be about 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 microns, or a number or range between any two of these values. In some embodiments, the center-to-center distance between the holes 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 microns. In some embodiments, the center-to-center distance between the wells can be about 4,890 microns.

[0115] The distance or spacing between the edges of the micropores can vary from about 1 micron to about 1,000 microns. In some embodiments, the distance between the edges of the holes can be or be about 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 microns, or a number or range between any two of these values. In some embodiments, the distance between the edges of the holes 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 microns. In some embodiments, the distance between the edges of the wells can be about 80 microns.

[0116] The microwell array can comprise microwells at varying densities, for example ranging from 100 microwells per square inch to 1,000,000 microwells per square inch. In some embodiments, the microwell array can have a density of or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, or about 100, 200, 300, 400, 500, 600, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 microwells per square inch. 00,000, 300,000, 400,000, 500,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 80,000, 900,000, 100,000,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 80,000, 900,000, 100,000,000 microwells, or a number or range between any two of these values. In some embodiments, the density of the microwell array can be or is about 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 per square centimeter, or a number or range between any two of these values. 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 per square centimeter. In some cases, each microwell array comprises from 10,000 microwells / cm 2 Up to 60,000 micropores / cm 2 density, such as 20,000 micropores / cm 2 Up to 50,000 micropores / cm 2 , and includes 36,000 micropores / cm2 Up to 42,000 micropores / cm 2 .

[0117] The total number of microwells on the substrate can vary depending on the pattern and spacing of the wells and the overall size of the array. The number of microwells in the array can vary, for example, ranging from about 96 to about 1,000,000. In some embodiments, the number of microwells in the microarray can be or is about 96, 384, 1536, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000 000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 10000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 100000000, 10 8 , 10 9 , or a value or range between any two of these values. In some embodiments, the number of microwells in the microarray can 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, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 90000, 100000, 200000, 300000, 400000, 500000, 600000 0000, 700000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 10000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 100000000, 10 8 , 10 9In some embodiments, the number of microwells in the microwell array can be about 96. In some embodiments, the number of microwells can be about 150,000. In some cases, each microwell array comprises 100,000 to 500,000 microwells, such as 200,000 to 250,000 microwells, and including 250,000 to 300,000 microwells. In some cases, each microwell array comprises at least 220,000 microwells per lane.

[0118] The micropores of the micropore array can additionally have any suitable spacing, where the spacing describes the amount of separation between the micropores. For example, in some cases, the calculation of the spacing includes the pore cross-sectional area and the wall separation. In some cases, the spacing ranges from 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 including 40 μm to 100 μm. In some cases, the spacing of the micropores is 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 pore pitch of 49 μm (39 μm pore cross-sectional area + 10 μm wall separation).

[0119] The microwell array can include surface features between the microwells that are designed to help guide cells and solid supports into the wells and / or prevent them from settling on the surface between the wells. Non-limiting examples of suitable surface features include, but are not limited to, dome-shaped, ridge-shaped, or pointed surface features that surround the wells or span the surface between the wells.

[0120] The microwells can be made using one of a variety of fabrication techniques. Non-limiting examples of fabrication methods that can be used include: bulk micromachining techniques such as photolithography and wet chemical etching, plasma etching, or deep reactive ion etching; micromolding and microimprinting; laser micromachining; 3D printing or other direct write fabrication processes using curable materials; and similar techniques.

[0121] Micropore array can be made of a variety of substrate materials.The selection of material depends on the selection of manufacturing technology, and vice versa.The non-limiting examples of suitable materials include fused quartz, glass, polymer, such as agarose, gelatin, hydrogel, polydimethylsiloxane (PDMS) elastomer, polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), polyimide, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), epoxy resin, thiol-ene resin, metal or metal film (such as aluminum, stainless steel, copper, nickel, chromium and titanium) etc.Hydrophilic material can be the ideal material (such as, to enhance wettability and minimize the non-specific binding of cells and other biomaterials) for manufacturing micropore array.The hydrophobic material that can be processed or coated (such as, by oxygen plasma treatment or grafting polyethylene oxide surface layer) can be used for manufacturing micropore array. To facilitate capillary wicking / evacuation of trapped gas or bubbles in the device, it may be desirable to fabricate the microwell array using a porous hydrophilic material. The microwell array can be made from a single material. The microwell array can also comprise two or more different materials that can be bonded or mechanically joined together.

[0122] The substrate can have a variety of shapes and sizes. For example, the shape (or footprint) of the substrate used to make the microwell can be square, rectangular, circular, or irregular. Its size can be characterized by its width, length, and depth.

[0123] The thickness range that is used to make the substrate of micropore can be from about 0.1mm to about 10mm, even bigger.The thickness of micropore array substrate can be or be about 0.1,0.5,1,2,3,4,5,6,7,8,9,10mm, or the numeral or scope between any two in these values.The thickness of micropore array substrate can be at least or at most 10,9,8,7,6,5,4,3,2,1,0.5,0.1mm.The thickness of micropore array substrate can be about 1mm.The thickness of micropore array substrate can be any value in these scopes, for example, the thickness of micropore array substrate can be between about 0.2mm to about 9.5mm.

[0124] Various surface treatment and surface modification techniques can be used to alter the properties of the microwell array surface. Examples may include, but are not limited to, oxygen plasma treatment to render the surface of a hydrophobic material more hydrophilic, wet or dry etching techniques to smooth or roughen glass and silicon surfaces, adsorption or grafting of polyethylene oxide or other polymer layers (e.g., pluronic or bovine serum albumin) onto the substrate surface to render it more hydrophilic and less susceptible to nonspecific adsorption of biomolecules and cells, and the use of silane reactions to graft chemically reactive functional groups onto 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, the selective addition or activation of chemically reactive functional groups (e.g., primary amines or carboxyl groups) on the inner walls of the microwells can be used to covalently couple oligonucleotide probes, peptides, proteins, or other biomolecules to the walls of the microwells. The choice of surface treatment or surface modification employed may depend on the type of surface properties desired and / or the type of material from which the microwell array is made.

[0125] Figures 9A-9C Different depictions of the flow cell microwells within the cartridge of the present invention are shown. Figure 9A Depicts microwells 901 in a flow cell of a multi-microwell array flow cell cartridge according to certain embodiments. Figure 9A As shown, the microwell array comprises hexagonal microwells. Figure 9B Provided Figure 9A A more detailed depiction of the hexagonal micropores shown, and Figure 9C Depicts spacing measurement according to one embodiment of the present invention. Figure 9C As shown, the pore pitch is 49 μm (39 μm pore cross-sectional area + 10 μm wall separation).

[0126] Figure 10 Depicted is the flow of fluid 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 flows through elongated channels 1003 of the flow cell. The flow cell contains a microwell array, e.g. Figures 9A-9C The microwell array discussed in

[10] is shown. Single cells can be trapped within the microwells. Liquid path 1004 leads to outlet 1005, which is stepped and includes raised portions through which the liquid must pass before it can be discharged. This prevents siphoning of the liquid.

[0127] box

[0128] The cartridge may also contain components designed to form physical or chemical barriers to prevent diffusion of macromolecules (or increase their path length and diffusion time) to minimize cross-contamination between microwells. Examples of such barriers include, but are not limited to: serpentine channel patterns for delivering cells and solid supports (e.g., beads) to the microwell array; retractable platens or deformable membranes that are pressed into contact with the surface of the microwell array substrate during the lysis or incubation steps; the use of larger beads (e.g., Sephadex beads described previously) to block the openings of the microwells; or the release of an immiscible hydrophobic fluid from a reservoir within the cartridge during the lysis or incubation steps to effectively separate and partition each microwell in the array.

[0129] The box can be manufactured using various techniques and materials known to those skilled in the art. Generally speaking, the box will be manufactured into a series of independent components and then assembled using a variety of mechanical assembly or bonding techniques. Suitable manufacturing techniques include, but are not limited to, conventional machining, CNC machining, injection molding, thermoforming, and 3D printing. After the box components are manufactured, they can be mechanically assembled using screws, clamps, etc., or permanently bonded using various techniques (depending on the selection of materials used), such as by using thermal bonding / welding or various adhesives or adhesive films, including epoxy, acrylic, silicone, UV-curable, polyurethane, or cyanoacrylate adhesives.

[0130] The box components can be made of a variety of suitable materials, including but not limited to silicon, fused quartz, glass, various polymers such as polydimethylsiloxane (PDMS; 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 polytetrafluoroethylene (PTFE), metals (such as aluminum, stainless steel, copper, nickel, chromium and titanium), or any combination thereof.

[0131] The cassette may include an integrated micropump or other fluid-driven mechanism for controlling the flow of fluid through the device. Examples of suitable micropumps or fluid-driven mechanisms include, but are not limited to, electromechanically or pneumatically driven microsyringes or plunger mechanisms, pneumatically or externally piston-driven diaphragm pumps, pneumatically driven reagent bags or capsules, or electroosmotic pumps.

[0132] The cartridge may contain microvalves for partitioning pre-loaded reagents or controlling fluid flow through the device. Suitable microvalves include, but are not limited to, disposable "valves" made with meltable or dissolvable wax or polymer plugs, or pierceable polymer membranes; 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 micro gate valves.

[0133] The cartridge may include a vent to provide a path for trapped air or gas (e.g., CO2 or N2) to escape. The vent can be constructed according to a variety of techniques, for example, using a porous plug made of polydimethylsiloxane (PDMS) or other hydrophobic material that allows capillary wicking of air or gas but prevents water penetration.

[0134] The mechanical interface features of the cartridge can ensure easy removal and highly accurate and repeatable positioning of the cartridge relative to the instrument system. Suitable mechanical interface features include, but are not limited to, alignment pins, alignment guides, mechanical stops, etc. Mechanical design features can include pressure relief features for bringing external equipment (e.g., magnets or optical elements) into close proximity with the microwell array chamber.

[0135] The cartridge may include a temperature control element or thermal interface feature for interfacing with an external temperature control module. Examples of suitable temperature control elements include, but are not limited to, resistive heating elements, miniature infrared emitting light sources, Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, and the like. The thermal interface feature may be made of a material that is a good thermal conductor (e.g., copper, gold, silver, etc.) and may include one or more flat surfaces that enable good thermal contact with an external heating or cooling block.

[0136] The cartridge may include optical interface features for optical imaging or spectroscopic interrogation of the microwell array. The cartridge may include an optically transparent window, such as on the microwell substrate itself or on the side of the flow cell or microarray chamber opposite the microwell array, made of a material that meets the spectral requirements of the imaging or spectroscopic techniques used to probe the microwell array. Suitable optical window materials include, but are not limited to, glass, fused silica, polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin polymer (COP), or cycloolefin copolymer (COC).

[0137] Box carrier

[0138] Embodiments of the present invention may also include a carrier for the cartridge. The carrier can be used, for example, for transporting the cartridge. In some cases, the cartridge carrier is configured to image the contents of the cartridge / flow cell in any microwell lane during the experiment. In some cases, the cartridge comprises one or more motion racks configured to be moved between analyzers (e.g., BD Rhapsody TMIn some cases, the cartridge carrier (and the cartridge when placed in the cartridge carrier) is oriented within a scanner). In some such cases, the number of moving racks in the cartridge carrier can range from 1 to 5, and includes 2 to 4. In selected cases, the cartridge carrier has 3 moving racks. The cartridge carrier may include one or more release portions. The release portions can be used to install or remove the cartridge from the holder. In some cases, the cartridge carrier includes chamfers so that the cartridge can be placed in a single orientation in the carrier. In some versions, the cartridge carrier includes a drip vessel configured to isolate droplets flowing from the cartridge outlet.

[0139] The box carrier can be configured for a single lane box (e.g., the box described in U.S. Patent Nos. 10,634,691 and 11,061,043), or a multi-micropore array flow cell box of the present invention. In some cases, the box carrier can be configured for a single lane box. In this case, the box carrier can include a dripping vessel that is positioned to isolate the droplets flowing out of a single outlet of the single lane box. In other cases, the box carrier can be configured for a multi-micropore array flow cell box of the present invention. In this case, the box carrier can include a dripping vessel that is positioned to isolate the droplets flowing out of each outlet of the multi-micropore array flow cell box of the present invention. In other cases, the box carrier can be configured for both a single lane box and a multi-micropore array flow cell box of the present invention. In some cases, the box carrier includes both a dripping vessel that is positioned to isolate the droplets flowing out of each outlet of the multi-micropore array flow cell box of the present invention, and a dripping vessel that is positioned to isolate the droplets flowing out of a single outlet of the single lane box.

[0140] The cartridge carrier can be made of any suitable material. In some cases, the cartridge carrier is constructed of a metal 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 some versions, the cartridge carrier is constructed of 6061-T6 aluminum alloy, hard anodized, and impregnated with Grade III PTFE.

[0141] The cassette carrier of the present invention is as follows Figures 11A-11H shown. Figures 11A-11B Different side profile views of the cartridge carrier 1100 are shown. Figure 11C A top view of the cartridge carrier 1100 is shown. Figure 11C As shown, cartridge 1100 includes a release portion 1101 configured to mount or remove the cartridge from a holder. Furthermore, cartridge 1100 includes drip vessels 1102a-b. Drip vessel 1102a is positioned to isolate droplets emanating from a single outlet of a single-lane cartridge, while drip vessel 1102b is positioned to isolate droplets emanating from each outlet of a multi-microwell array flow cell cartridge of the present invention. Also shown are cartridge offset release portion 1103 and chamfer 1104. Figure 11DA bottom view of the cartridge holder 1100 is shown, with the motion frame 1105 visible. Figures 11E-11F A 45° angle view of the cartridge carrier 1100 is shown, and Figures 11G-11H A side view of the cartridge carrier 1100 is shown.

[0142] Sample collection container holder

[0143] As described above, aspects of the present invention also include a sample collection container holder. The sample collection container holder of interest is configured to receive a plurality of sample collection containers for collecting analytes from a multi-microwell array flow cell box (e.g., as described above). The sample collection container holder can be configured to accommodate any suitable number of sample collection containers, e.g., the number of sample collection containers ranges from 2 to 10, including 7 to 9. In some cases, the system comprises 8 sample collection containers. In selected embodiments, the sample collection container holder includes a counterweight that is configured to maintain the sample collection container in an upright position. In certain embodiments, the shape of the sample collection container holder complements the shape of the cell analysis system so that the sample collection container holder can be received in the cell analysis system in a single orientation. In other words, the sample collection container holder can have a mistake-proofing design. In some cases, the sample collection container holder has a rounded end.

[0144] The sample container holder can be constructed of a variety of suitable materials, including but not limited to silicon, fused quartz, glass, various 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 polytetrafluoroethylene (PTFE), metals (such as aluminum, stainless steel, copper, nickel, chromium and titanium), or any combination thereof.

[0145] Figures 12A-12C FIG. 2 shows a sample collection container holder according to certain embodiments of the present invention. Figure 12A As shown, the sample collection container rack 1201 is configured to accommodate eight sample collection containers 1202a-1202h, which are depicted as test tubes. Figure 12B A top view of the sample collection container holder 1201 is shown. As shown by its rounded ends, the shape of the sample collection container holder 1201 allows it to be inserted into the drawer of the system of the present invention in only one orientation. Figure 12C As shown, the sample collection container holder 1201 includes a counterweight 1203 configured to maintain the sample collection container holder in an upright position.

[0146] Methods for processing cell samples

[0147] As described above, aspects of the present invention include methods for processing cell samples. The method of interest includes introducing a multi-microwell array flow cell cartridge into a system comprising a tray configured to receive the multi-microwell array flow cell cartridge, and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell of the multi-microwell array flow cell cartridge when in an enabled position. The method also includes loading a sample into the multi-microwell array flow cell cartridge, driving the retrieval magnet assembly to an enabled position, and applying a uniform magnetic force to the sample within the multi-microwell array flow cell cartridge to produce a processed sample, and collecting the processed sample from the multi-microwell array flow cell cartridge.

[0148] In some cases, the method includes loading a plurality of different samples into different flow cells of the box. The number of samples can vary, and in some cases, can range from 2 to 10 (e.g., 8). The method according to certain embodiments includes loading the lysis buffer into the porous box after the sample liquid is loaded into the porous box. In some versions, the method includes loading the lysis buffer into the porous box without using a lysis magnet. The method according to a selected form of the present invention includes loading the barcoded beads into the porous box before driving the retrieval magnet assembly. In some cases, the barcoded beads may comprise a nucleic acid barcode comprising a universal primer binding domain, a cell marker domain, and a target capture domain. In selected embodiments, the target capture domain is a poly (T) sequence. In some cases, the nucleic acid barcode also comprises a unique molecular index (UMI).

[0149] In some cases, the method includes applying a uniform magnetic field from a position above the tray. As described above, a system that can be used for the subject method includes: a tray configured to accommodate a multi-microwell array flow cell cartridge; and a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cell in the cartridge when in the enabled position. In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force from a position above the tray. In some cases, the uniform magnetic force can be a magnetic field in the range of 650 gauss to 1325 gauss. In some cases, the retrieval magnet assembly is configured to apply a uniform magnetic force via a plurality of magnets. In some such cases, the plurality of magnets have alternating polarity. The number of magnets in the retrieval magnet assembly can vary, and in some cases ranges from 1 to 10 (e.g., 4). The type of magnet in the retrieval magnet assembly can 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 can 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 system of the present invention can be configured to adjust the position of the retrieval magnet assembly to process the cell sample. In some embodiments, the retrieval magnet assembly is drivable between an activated position and a deactivated position, wherein the activated position is located near the tray and the deactivated position is located at a greater distance from the tray relative to the activated position.

[0150] In some forms, the method includes using a sample collection container support, which is configured to receive multiple sample collection containers, which are used to collect analytes from the box. In some such forms, the sample collection container support includes a counterweight, which is configured to maintain the sample collection support in an upright position. The system and the sample collection container support may also have complementary shapes so that the sample collection container support 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 can vary, and in some cases can be 2 to 10 (e.g., 8). In some cases, the number of sample collection containers included in the system matches the number of flow cells in the box. In some embodiments, the system of the present invention includes a waste collection container for collecting liquid waste from a multi-microporous array flow cell box. In some cases, the method includes an interlocking device, which is configured to prevent sample liquid from being collected into the waste collection container when the retrieval magnet assembly is in an enabled position. In some cases, the tray includes a latch for holding the multi-microporous array flow cell box. In the selected form, the system does not include a lysis magnet in the position below the tray.

[0151] As described above, the method of the present invention includes the use of a multi-micropore array flow cell box. The multi-micropore array flow cell box used for this method includes a plurality of fluid lanes, each fluid lane including an inlet for receiving liquid, a flow cell including a micropore array, and an outlet for discharging liquid. In some cases, the multi-micropore array flow cell box includes a number ranging from 2 to 10 (e.g., 8) fluid lanes. In some forms, each outlet is stepped to prevent siphoning liquid from the flow cell. In some embodiments, each flow cell includes an elongated channel. The length of the elongated channel can vary, and in some cases can be in the range of 50 mm to 100 mm. The number of micropores in each micropore array can also vary, and in some cases can be in the range of 250,000 micropores to 300,000 micropores. The micropore density within each micropore can vary, and in some cases, can range from 36,000 micropores / cm 2 Up to 42,000 micropores / cm 2 .

[0152] The type of analysis performed in the subject method can vary as needed. In some cases, the method includes generating a sequencing-ready nucleic acid library from the processed sample. In certain embodiments, the sequencing-ready nucleic acid library can be sequenced using a next-generation sequencing protocol. In other embodiments, the method is a method for genomic analysis, epigenomic analysis, transcriptomic analysis, or proteomic analysis. In some forms, the method is a method for multi-omic analysis, for example, wherein the multi-omic analysis comprises at least transcriptomic and proteomic analysis.

[0153] Random barcode

[0154] In some embodiments, the subject method includes barcoding (e.g., random barcoding). Random barcoding has been described in, for example, US20150299784, WO2015031691, and Fu et al., Proc Natl Acad Sci U. SA 2011 May 31; 108(22): 9026-31, the contents of which are incorporated herein in their entirety. In short, a random barcode can be a polynucleotide sequence that can be used for random labeling (e.g., barcode, tag) targets. A random barcode can include one or more tags. Exemplary tags can include universal tags, cell tags, molecular tags, sample tags, well plate tags, spatial tags, and / or pre-space tags. A random barcode can include a 5' amine that can connect the random barcode to a solid support. A random barcode can include universal tags, dimensional tags, spatial tags, cell tags, and / or molecular tags. The order of different tags (including but not limited to universal tags, dimensional tags, spatial tags, cell tags, and molecular tags) in a random barcode can vary. For example, the universal marker can be the most 5' end marker, while the molecular marker can be the most 3' end marker. The spatial marker, dimensional marker, and cell marker can be arranged in any order. In some embodiments, the universal marker, spatial marker, dimensional marker, cell marker, and molecular marker are arranged in any order.

[0155] The random barcodes can come from a "non-depletable reservoir", that is, a pool of random barcodes consisting of many different tags. The non-depletable reservoir can contain a large number of different random barcodes, so that when the non-depletable reservoir is associated with a pool of targets, each target can be associated with a unique random barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the ratio of the number of copies of the same target molecule in the set to the diversity of the tags. The size of the resulting set of labeled target molecules can be determined by the randomness of the barcoding process, and the number of target molecules present in the original set or sample can then be calculated by analyzing the number of random barcodes detected. When the ratio of the number of copies of the target molecules present to the number of unique random barcodes is low, the labeled target molecules have a high degree of uniqueness (i.e., the probability of more than one target molecule being labeled by a given tag is very low).

[0156] Mark, such as cell marker, can comprise a group of unique, length-determined nucleic acid subsequences, such as each subsequence comprises seven nucleotides (equivalent to the number of digits used in some Hamming codes (Hamming error correction code), and these subsequences can be designed to provide error correction capabilities. This group of error correction subsequences comprises seven nucleotide sequences, and its design makes any paired sequence combination in this group show determined " genetic distance " (or mismatch base number), such as, one group of error correction subsequences can be designed to have a genetic distance of three nucleotides. In this case, the error correction sequence (hereinafter described in more detail) in the sequence data set of the marker target nucleic acid molecule can be allowed to detect or correct amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequence for generating error correction code can vary, such as, their length can be or be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or the number or scope between any two of these values. In some embodiments, nucleic acid subsequences of other lengths can be used to generate error correction code.

[0157] The random barcode can include a target binding region. The target binding region can interact with a target in the sample. The target can be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each including a poly (A) tail, and any combination thereof. In some embodiments, multiple targets can include deoxyribonucleic acid (DNA).

[0158] In some embodiments, the target binding region may comprise an oligo (dT) sequence that can interact with the poly (A) tail of the mRNA. One or more markers of the random barcode (e.g., universal markers, dimensional markers, spatial markers, cell markers, and molecular markers) may be separated from the remaining one or two markers of the random barcode by a spacer. The spacer may 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 markers of the random barcode are separated by a spacer.

[0159] The random barcode may comprise one or more universal markers, one or more dimensional markers, one or more spatial markers, one or more cellular markers, one or more molecular markers, one or more target binding regions, or any combination thereof.

[0160] For all random barcodes in the random barcode set attached to a given solid support (e.g., beads), one or more universal tags may be identical, or for all random barcodes attached to a plurality of beads, one or more universal tags may be identical. The universal tag may comprise a nucleic acid sequence that can hybridize with a sequencing primer. The universal tag may comprise a nucleic acid sequence that can hybridize with a PCR primer, or comprise a nucleic acid sequence that can hybridize with a sequencing primer and a PCR primer. The nucleic acid sequence of the universal tag that can hybridize with a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal tag may comprise a sequence that can be used to start transcription of a random barcode. The universal tag may comprise a sequence that can be used to extend the random barcode or the region within the random barcode.

[0161] Dimensional tags can include nucleic acid sequences that provide information about the dimensions in which random labeling occurs. For example, dimensional tags can provide information about the time when a target is randomly barcoded. Dimensional tags can be associated with the time of random barcoding in a sample. Dimensional tags can be activated at the time of random labeling. Different dimensional tags can be activated at different times. Dimensional tags provide information about the order in which targets, target groups, and / or samples are randomly barcoded. For example, a cell population can be randomly barcoded in the G0 phase of the cell cycle. Cells can be pulsed with random barcodes again in the G1 phase of the cell cycle. Cells can be pulsed with random barcodes again in the S phase of the cell cycle, and so on. The random barcodes of each pulse (e.g., each stage of the cell cycle) can contain different dimensional tags. In this way, dimensional tags provide information about which targets are labeled at which stage of the cell cycle. Dimensional tags can query many different biological times. Exemplary biological times can include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, samples (e.g., cells, cell populations) can be stochastically labeled before and / or after treatment with a drug and / or therapy. Changes in the copy number of different targets can indicate the response of the sample to the drug and / or therapy.

[0162] Solid support

[0163] In some embodiments, the random 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 markers (for example, a first molecular marker) of a plurality of random barcodes (for example, a first group of multiple random barcodes) on a solid support differ by at least one nucleotide. The cell markers of the random barcodes on the same solid support can be the same. The cell markers of the random barcodes on different solid supports can differ by at least one nucleotide. For example, the first cell marker of the first multiple random barcodes on the first solid support can have the same sequence, and the second cell marker of the second multiple random barcodes on the second solid support can have the same sequence. The first cell marker of the first multiple random barcodes on the first solid support and the second cell marker of the second multiple random barcodes on the second solid support can differ by at least one nucleotide. The length of the cell marker can be, for example, about 5-20 nucleotides. The length of the molecular marker can be, for example, about 5-20 nucleotides.

[0164] The synthetic particles can be, for example, beads. The beads can be, for example, silica beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can comprise the following materials: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0165] For example, after cells (e.g., single cells) are introduced into a plurality of microwells of a microwell array, beads can be introduced into a plurality of microwells of the microwell array. Each microwell can contain a bead. The beads can contain a plurality of random barcodes. The random barcodes can include a 5' amine region attached to the beads. The random barcodes can include universal markers, molecular markers, target binding regions, or any combination thereof.

[0166] The random barcodes disclosed herein can be associated with a solid support (e.g., a bead) (e.g., attached to a solid support). Each of the random barcodes associated with a solid support can include a molecular marker selected from at least 100 or 1000 molecular markers with a unique sequence. In some embodiments, different random barcodes associated with a solid support can include molecular markers of different sequences. In some embodiments, a certain percentage of the random barcodes associated with a solid support include the same cell marker. For example, the percentage can be or be about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a numerical value or range between any two of these values. For 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 random barcodes associated with a solid support can have the same cell marker. The random barcodes associated with different solid supports can have different cell markers selected from at least 100 or 1000 cell markers having unique sequences.

[0167] In some cases, the barcode microbeads contain a nucleic acid barcode that includes a universal primer binding domain, a cell marker domain, and a target capture domain. In selected cases, the target capture domain is a poly(T) sequence. In some versions, the nucleic acid barcode also contains a unique molecular index (UMI). Random barcodes with unique molecular labels (also called molecular indices (MI)) can be used to count the number of molecules and correct for amplification bias. TM Random barcoding, such as the assay (Cellular Research, Inc. (Palo Alto, Calif.)), can correct for biases caused by PCR and library preparation steps by labeling mRNA with molecular markers (MLs) during reverse transcription (RT). In some embodiments, the method includes using a non-depleting pool of random barcodes with a large number (e.g., 6561 to 65536) of unique molecular markers on poly (T) oligonucleotides to hybridize with all poly (A)-mRNA in the sample during the RT step. The random barcodes can include universal PCR priming sites. During the RT process, target gene molecules react randomly with the random barcodes. Each target molecule can hybridize with the random barcodes, thereby generating a randomly barcoded complementary ribonucleotide (cDNA) molecule. After labeling, the randomly barcoded cDNA molecules from the microwells of a microplate can be combined into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to generate the number of reads, the number of random barcodes with unique molecular markers, and the number of mRNA molecules.

[0168] In some embodiments, a solid support comprising a plurality of synthetic particles associated with a plurality of random barcodes can be used to stochastically barcode a plurality of targets in a sample. In some embodiments, the solid support can comprise a plurality of synthetic particles associated with a plurality of random barcodes. The spatial labels of the plurality of random barcodes on different solid supports can differ by at least one nucleotide. For example, the solid support can comprise a plurality of random barcodes in two or three dimensions. The synthetic particles can be beads. The beads can be silica beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support can comprise a polymer, a substrate, a hydrogel, a needle array device, an 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 random barcodes can be unassociated with the solid support. The random barcodes can be individual nucleotides. The random barcodes can be associated with a substrate.

[0169] As used herein, the terms "tethered," "attached," and "immobilized" are used interchangeably to refer to the covalent or non-covalent attachment of random barcodes to a solid support. A variety of different solid supports can be used as solid supports for attaching presynthesized random barcodes or in situ solid phase synthesized random barcodes.

[0170] In some embodiments, the solid support is a bead. The bead can include one or more types of solid, porous or hollow spheres, spheres, bearings, cylinders or other similar structures, and the nucleic acid can be fixed thereto in a covalent or non-covalent manner. The bead can be composed of, for example, plastic, ceramic, metal, polymeric material or any combination thereof. The bead can be (or include) independent particles of spherical shape (e.g., microspheres), and also has a non-spherical or irregular shape, such as a cube, a cuboid, a pyramid, a cylinder, a cone, an ellipse (oblong) or a disc, etc. In some embodiments, the bead can be non-spherical.

[0171] 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 (Fe3O4; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, certain alloys thereof, and certain rare earth metal compounds), ceramics, plastics, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, and any combination thereof. In some embodiments, the beads (e.g., beads to which the random labels are attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0172] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 microns to 50 microns. In some embodiments, the diameter of the beads can be or is about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 microns, or a value or range between any two of these values.

[0173] The diameter of the beads can be related to the diameter of the substrate pores. In some embodiments, the diameter of the beads can be longer or smaller than the pore diameter or approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% longer or smaller than the pore diameter, or a numerical value or range between any two of these values. The diameter of the beads can be related to the diameter of the cells (e.g., a single cell captured by the substrate pores). In some embodiments, the diameter of the beads can be longer or smaller than the cell diameter or approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% longer or smaller than the cell diameter, or a numerical value or range between any two of these values.

[0174] 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 in the substrate (e.g., gel, substrate, framework, or polymer) can be identified by spatial markings on the random barcode on the beads, which can serve as location addresses.

[0175] Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, 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-fluorochrome microbeads, and BcMag TM Carboxyl-terminated magnetic beads.

[0176] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes, causing the beads to fluoresce in one or more fluorescent channels. The beads can be associated with iron oxide or chromium oxide, causing them to be paramagnetic or ferromagnetic. The beads can be identifiable. 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 contain a random barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible.

[0177] The solid support (e.g., bead) can be visualized. The solid support can include a visualization label (e.g., a fluorescent dye). The solid support (e.g., bead) can be etched with an identifier (e.g., a number). The identifier can be visualized by imaging the bead.

[0178] Random barcoding method

[0179] Provided herein is a method for estimating the number of different targets at different locations in a physical sample (e.g., tissue, organ, tumor, cell). The method may include placing a random barcode in close proximity to the sample, cracking the sample, associating different targets with the random barcode, amplifying the target, and / or performing digital counting on the target. The method may also include analyzing and / or visualizing the information obtained from the spatial tags on the random barcode. In some embodiments, the method includes visualizing multiple targets in the sample. Mapping multiple targets to the sample map may include generating a two-dimensional map or a three-dimensional map of the sample. The two-dimensional map and the three-dimensional map may be generated before or after random barcoding of the multiple targets in the sample. Visualizing multiple targets in the sample may include mapping multiple targets to the sample map. Mapping multiple targets to the sample map may include generating a two-dimensional map or a three-dimensional map of the sample. The two-dimensional map and the three-dimensional map may be generated before or after random barcoding of the multiple targets in the sample. In some embodiments, the two-dimensional map and the three-dimensional map may be generated before or after cracking the sample. Lysing the sample before or after generating the two-dimensional or three-dimensional map may include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0180] In some embodiments, stochastically barcoding the plurality of targets comprises hybridizing the plurality of random barcodes to the plurality of targets to generate stochastically barcoded targets. Stochastically barcoding the plurality of targets can comprise generating an indexed library of stochastically barcoded targets. Generating the indexed library of stochastically barcoded targets can be performed using a solid support comprising a plurality of random barcodes.

[0181] The present disclosure provides a method for contacting a sample (e.g., a cell) with a substrate of the present disclosure. A sample comprising, for example, a cell, an organ, or a tissue thin section can be contacted with a random barcode. Cells can be contacted, for example, by gravity flow, where the cells can settle and form a monolayer. The sample can be a tissue thin section. The thin section can be placed on a substrate. The sample can be one-dimensional (e.g., forming a plane). The sample (e.g., a cell) can be diffused onto the entire substrate, for example, by growing / culturing cells on a substrate.

[0182] When the random barcode is in close proximity to the target, the target can hybridize with the random barcode. The random barcodes can be contacted at a non-depleting ratio so that each different target can associate with a different random barcode of the present disclosure. To ensure effective association between the target and the random barcode, the target can be cross-linked to the random barcode.

[0183] Cell lysis

[0184] As cells and random barcodes are distributed, cells can be cracked to release target molecules. Cell lysis can be accomplished in a variety of ways, such as by chemical or biochemical methods, by osmotic shock, or by thermal cracking, mechanical cracking, or photolysis. Cells can be lysed by adding a cell lysis buffer comprising a detergent (e.g., SDS, lithium lauryl 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. In order to increase the association of the target with the random barcode, the diffusion rate of the target molecule can be changed, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0185] In some embodiments, filter paper can be used to lyse the sample. The filter paper can be soaked with lysis buffer on top of the filter paper. The filter paper can be applied to the sample under pressure, which can promote sample lysis and hybridization of the sample target with the substrate.

[0186] In some embodiments, the lysis can be carried out by mechanical lysis, thermal lysis, photolysis and / or chemical lysis. Chemical lysis can include the use of digestive enzymes, such as proteinase K, pepsin and trypsin. The lysis can be carried out by adding a lysis buffer to the substrate. The lysis buffer can comprise Tris HCl. The lysis buffer can comprise at least about 0.01, 0.05, 0.1, 0.5 or 1M or more Tris HCl. The lysis buffer can comprise up to about 0.01, 0.05, 0.1, 0.5 or 1M or more Tris HCl. The lysis buffer can comprise about 0.1M 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 higher. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or higher. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). The concentration of salt in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more. The concentration of salt in the lysis buffer can be at most about 0.1, 0.5, or 1 M or more. In some embodiments, the concentration of salt in the lysis buffer is about 0.5 M. The lysis buffer can contain a detergent (e.g., SDS, lithium dodecyl sulfate, triton X, tween, NP-40). The concentration of 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 the 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 the detergent in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent is used, the shorter the time required for lysis. The lysis buffer can contain 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 may contain a reducing agent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or higher. The concentration of the reducing agent in the lysis buffer may be at most about 1, 5, 10, 15, or 20 mM or higher.In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM.In some embodiments, the lysis buffer can comprise 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.

[0187] Lysis can be carried out at a temperature of about 4, 10, 15, 20, 25 or 30° C. Lysis can be carried out for about 1, 5, 10, 15 or 20 minutes or longer. The 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. The 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.

[0188] Attachment of random barcodes to target nucleic acid molecules

[0189] After the cells are lysed and the nucleic acid molecules are released therefrom, the nucleic acid molecules can be randomly associated with the random barcodes of the co-located solid support. The association can include hybridization of the target recognition region of the random barcode with the complementary portion of the target nucleic acid molecule (for example, the oligo (dT) of the random barcode can interact with the poly (A) tail of the target). The assay conditions for hybridization (for example, buffer pH, ionic strength, temperature, etc.) 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 multiple probes on the substrate (for example, hybridized with the probes on the substrate). When the probe contains oligo (dT), the mRNA molecule can hybridize with the probe and be reverse transcribed. The oligo (dT) portion of the oligonucleotide can serve as a primer for the first chain synthesis of the cDNA molecule. For example, the mRNA molecule can hybridize with the random barcode on the bead. For example, a single-stranded nucleotide fragment can hybridize with the target binding region of the random barcode.

[0190] Attachment can also include connecting the target recognition region of random barcode to the part of target nucleic acid molecule.For example, the target binding region can include a nucleic acid sequence that can specifically hybridize with a restriction site overhang (for example, EcoRI sticky end overhang). The assay procedure can also include processing the target nucleic acid to produce a restriction site overhang with a restriction endonuclease (for example, EcoRI). Then, the random barcode can be connected to any nucleic acid molecule that comprises a complementary sequence to the restriction site overhang. Two fragments can be joined using a ligase (for example, T4 DNA ligase).

[0191] For example, labeled targets (e.g., target barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., into a tube. Pooling of labeled targets can be accomplished by, e.g., retrieving magnetic beads to which random barcodes and / or target barcode molecules are attached.

[0192] Retrieval of a solid support-based collection of attached target barcode molecules can be performed using magnetic beads and an externally applied magnetic field. After the target barcode molecules are pooled, all further processing can be performed in 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 the microwells, that is, without first pooling labeled target nucleic acid molecules from multiple cells.

[0193] Figure 13 A non-limiting example of a workflow 1300 that can be performed using the system and multi-microwell array flow cell cartridge of the present invention is depicted. Workflow 1300 describes the steps performed after the multi-microwell array flow cell cartridge is placed in a tray and the sample collection container is placed in a drawer. If these components are not already in place, additional steps may be required to insert the cartridge into the tray and / or insert the sample collection container or sample collection container holder before initiating workflow 1300.

[0194] Additionally, workflow 1300 describes steps performed when the retrieval magnet assembly drive is in the deactivated position and the drawer drive is in the waste collection position at the start of workflow 1300. One skilled in the art will appreciate that additional steps may be required to move the drive to initiate the workflow.

[0195] Workflow 1300 can start from step 1301, wherein multiple cells are introduced into one or more fluid swimming lanes of box, and box is positioned in system of the present invention simultaneously.In some embodiments, multiple cells can be introduced into box via entrance (for example, gasket).In some embodiments, multiple cells can be introduced into box via pipette.In some embodiments, multiple cells can enter micropore array via the flow pool of box.In some embodiments, each micropore in micropore array can only capture single cell in multiple cells.In alternative embodiment, multiple cells can be introduced into micropore array before box is positioned in system.

[0196] After the cells are introduced into the microwell array, in step 1302, a plurality of barcoded beads can be introduced into the microwell array. In some embodiments, a plurality of beads can be introduced into the box inlet. In some embodiments, a plurality of beads can be introduced into the box via a pipette. In some embodiments, a plurality of beads can be entered into the microwell array via the flow pool of the box. In an alternative embodiment, a plurality of beads can be introduced into the microwell array before the box is positioned to the system of the present invention. It will be understood by those skilled in the art that there is no particular limitation on the order of introducing cells (frame 1301) and introducing beads (frame 1302). These 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 only capture a single bead in a plurality of beads. In some embodiments, each microwell in the microwell array can capture a single cell in a plurality of cells and a single bead in a plurality of beads.

[0197] After the plurality of beads are 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 are introduced into the microwell array. Cell lysis can be accomplished by any of the methods described herein. In some embodiments, step 1303 does not include activating a lysis magnet. In some embodiments, the size of the beads can be designed such that the beads located above the cells in the microwells can prevent the cells from flowing out of the microwells without removing the beads from the microwells.

[0198] In some embodiments, lysis is performed by introducing a lysis buffer. In some embodiments, during the cell lysis process, a drawer drive can be used to position the drawer so that a waste collection container is located below the cartridge outlet to receive excess buffer flowing through the flow cell. In some embodiments, the introduction of cells and beads results in the cells and / or beads being located within the flow cell but outside the microwells. In these embodiments, cells and / or beads located outside the microwells may be washed away by the lysis buffer and into the waste collection container.

[0199] In certain embodiments, washing can be performed before, during, or after cell lysis. In some embodiments, a washing solution can be introduced into the flow cell of the cartridge via an inlet. The washing solution can flow through the flow cell to remove beads and / or cells that are within the flow cell but outside the micropores. The removed beads and / or cells can be deposited in a waste collection container aligned with the flow cell outlet.

[0200] After cell lysis, the barcoded beads can be retrieved in step 1304. In some embodiments, the barcoded beads are retrieved by advancing the retrieval magnet assembly from its inactive position to its active position. As described herein, the retrieval magnet assembly can be advanced from its inactive position to its active position by movement of a retrieval magnet assembly driver.

[0201] When the retrieval magnet assembly is in the enabled position, the retrieval magnet assembly can attract the barcoded beads located in the micropores. In some embodiments, the uniform magnetic force applied by the retrieval magnet assembly to the barcoded beads can be sufficient to remove the barcoded beads from the micropores. When the barcoded beads are removed by the retrieval magnet assembly, cells may still remain in the micropores. In some embodiments, the magnetic force applied by the retrieval magnet assembly to the barcoded beads can move the barcoded beads toward the upper surface of the box. The magnetic force applied by the retrieval magnet assembly can maintain the magnetic beads at a position above the micropores. When the magnetic beads are maintained at a position above the micropores, the magnetic beads can be considered to have been retrieved by the retrieval magnet assembly.

[0202] When the retrieval magnet assembly maintains the magnetic beads at a position higher than the micropores, washing can be performed. In some embodiments, a wash solution can be introduced into the flow cell of the cartridge via the inlet. The wash solution can flow through the flow cell to remove cells in the micropores. The cells can be deposited in a waste collection container aligned with the flow cell outlet. Washing cells from the micropores can allow only the beads previously placed in the micropores to be collected later. In some embodiments, actuation of the retrieval magnet assembly causes an interlocking device to engage with the drawer, thereby preventing the drawer from moving.

[0203] After the beads are retrieved by the retrieval magnet assembly, they can be collected in step 1305. In some embodiments, the retrieval magnet assembly is switched from its activated position to its deactivated position, thereby releasing the beads from their holding position above the microwell. As described herein, the retrieval magnet assembly can be switched from its activated position to its deactivated position by movement of a retrieval magnet assembly driver. In some embodiments, the beads, after being released, may fall or return to the same microwell from which they were removed.

[0204] After releasing the beads, the sample collection container in the sample collection holder can be aligned with the outlet of the box. As described herein, by switching the drive to the sample collection position, the sample collection container in the sample collection holder can be aligned with the outlet. After alignment, the fluid can be pushed into the flow cell so that the beads flow out of the outlet and into the sample collection container. In order to collect the beads, it may be desirable to place the retrieval magnet assembly in its inactive position. After collecting the beads, workflow 1300 ends. In some embodiments, after collecting the beads, the sample collection container can be removed for further processing and / or analysis.

[0205] Library preparation

[0206] In some cases, the method further includes a sequence library preparation protocol for further processing the analyte (e.g., DNA, RNA) obtained by the above steps to generate a sequence library (e.g., a cDNA library). In some cases, the method includes generating a sequencing-ready nucleic acid library from the processed sample. In some versions, the sequencing-ready nucleic acid library can be sequenced using a next-generation sequencing (NGS) protocol.

[0207] In some embodiments, barcoding multiple targets in a sample (e.g., random barcoding) further comprises generating an index library of barcoded targets (e.g., randomly barcoded targets) or barcoded target fragments. The establishment of an index library is discussed in, for example, U.S. Patent No. 10,676,779 and U.S. Patent Application Publication No. 2021 / 0171940; the disclosures of which are incorporated herein by reference in their entirety. The barcode sequences of different barcodes (e.g., molecular markers of different random barcodes) can be different from each other. Generating an index library of barcoded targets comprises generating multiple index polynucleotides from multiple targets in a sample. For example, for an index library of barcoded targets comprising a first index target and a second index target, the tag region of the first index polynucleotide can differ from the tag region of the second index polynucleotide by about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between any two of these values. In some embodiments, generating an index 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 tag region; and performing first-strand synthesis using a reverse transcriptase to generate single-stranded labeled cDNA molecules, each cDNA molecule comprising a cDNA region and a tag 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 index library of barcoded targets can also comprise amplifying the single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules; and performing nested PCR on the double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method can include generating adaptor-tagged amplicons.

[0208] Barcoding (e.g., random barcoding) can include labeling individual nucleic acid (e.g., DNA or RNA) molecules using nucleic acid barcodes or tags. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters can be added for sequencing, such as using next-generation sequencing (NGS). Sequencing results can be used to determine cell markers, molecular markers, and nucleotide fragment sequences for one or more target copies.

[0209] Figure 14A A workflow for practicing one embodiment of the subject method is shown. As shown in step 1451, cells and magnetic beads are loaded into a cartridge of the present invention. Within a microwell, one cell is paired with one barcoded magnetic bead. In step 1452, the cells are lysed, and nucleic acids from the cells are hybridized to the magnetic beads. In step 1453, the magnetic beads are retrieved using a retrieval magnet assembly. In step 1454, cDNA is synthesized using the nucleic acids.

[0210] Figure 14B is a schematic diagram showing a non-limiting example process for generating an indexed library of barcoded targets (e.g., randomly barcoded targets), such as barcoded mRNAs or fragments thereof. In other words, Figure 14B Shows Figure 14A 14. The example cDNA synthesis step 1454 shown in FIG. As shown in step 1, the reverse transcription process can encode unique molecular markers, cell markers, and universal PCR sites for each mRNA molecule. Specifically, by hybridizing (e.g., random hybridization) a set of barcodes (e.g., random barcodes) 1410 with a poly (A) tail region 1408 of an RNA molecule 1402, the RNA molecule 1402 can be reverse transcribed to produce a labeled cDNA molecule 1404, including a cDNA region 1406. Each barcode 1410 can include a target binding region, such as a poly (dT) region 1412, a marker region 1414 (e.g., a barcode sequence or molecule), and a universal PCR region 1416. In some embodiments, the cell marker can include 3 to 20 nucleotides. In some embodiments, the molecular marker can include 3 to 20 nucleotides. In some embodiments, each of the plurality of random barcodes further comprises one or more universal markers and cell markers, wherein the universal markers of the plurality of random barcodes on the solid support are the same, and the cell markers of the plurality of random barcodes on the solid support are the same. In some embodiments, the universal marker may comprise 3 to 20 nucleotides. In some embodiments, the cell marker comprises 3 to 20 nucleotides.

[0211] In some embodiments, the label region 1414 may include a barcode sequence or molecular marker 1418 and a cell marker 1420. In some embodiments, the label region 1414 may include one or more universal markers, dimensional markers, and cell markers. The length of the barcode sequence or molecular marker 1418 can be, can be about, can be at least, can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range between any of these values. The length of the cell marker 1420 can be, can be about, can be at least, can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range between any of these values. The length of the universal tag can be, can be about, can be at least, can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range between any of these values. The universal tag can be the same for multiple random barcodes on the solid support, and the cell tags can also be the same for multiple random barcodes on the solid support. The length of the dimensional tag can be, can be about, can be at least, can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range between any of these values.

[0212] In some embodiments, the marker region 1414 can comprise, comprise about, comprise at least, or comprise 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 different markers (e.g., barcode sequences or molecular markers 1418 and cellular markers 1420), or a number or range between any of these values. Each marker can be, can be about, can be at least, or can be at most 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. The barcode or random barcode set 1410 may include, include about, include at least, or at most 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 108 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 The barcodes or random barcodes 1410 may be a number or range between any of these values. Furthermore, the barcodes or random barcodes 1410 may each contain a unique tag region 1414, for example. The labeled cDNA molecules 1404 may be purified to remove excess barcodes or random barcodes 1410. Purification may include Ampure bead purification.

[0213] As shown in step 2, the products from the reverse transcription process in step 1 can be combined into one tube and PCR amplified using the first PCR primer pool and the first universal PCR primer. The combination is possible due to the unique tag region 1414. Specifically, the tagged cDNA molecules 1404 can be amplified to produce nested PCR tag amplicons 1422. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification of 96 multiplex primers within a single reaction volume. In some embodiments, the multiplex PCR amplification can utilize, utilize approximately, utilize at least, or utilize at most 10, 20, 40, 50, 70, 80, 90, 10 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 Multiplexed primers, or a number or range between any of these values. Amplification can use a first PCR primer pool 1424, which contains custom primers 1426A-C for a specific gene and a universal primer 1428. Custom primers 1426A-C can hybridize to regions within cDNA portion 1406' of labeled cDNA molecule 1404. Universal primer 1428 can hybridize to universal PCR region 1416 of labeled cDNA molecule 304.

[0214] like Figure 14BAs shown in step 3 of the method, the product 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. Specifically, the amplicons 1422 of the nested PCR markers can be further amplified by nested PCR. Nested PCR can include multiplex PCR using 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 nested PCR primer pool can include, comprise approximately, comprise at least, or comprise 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 different nested PCR primers 1430, or a number or range between any of these values. Nested PCR primer 1432 may include adapter 1434 and hybridize to a region within cDNA portion 1406″ of tagged amplicon 1422. Universal primer 1428′ may include adapter 1436 and hybridize to universal PCR region 1416 of tagged amplicon 1422. Thus, step 3 produces adapter-tagged amplicon 1438. In some embodiments, nested PCR primer 1432 and second universal PCR primer 1428′ may not include adapters 1434 and 1436. Adapters 1434 and 1436 may be ligated to the product of the nested PCR to produce adapter-tagged amplicon 1438.

[0215] As shown in step 4, the PCR product in step 3 can be PCR amplified using library amplification primers for sequencing. Specifically, adapters 1434 and 1436 can be used to perform one or more additional assays on adapter-tagged amplicon 1438. Adapters 1434 and 1436 can hybridize with primers 1440 and 1442. One or more primers 1440 and 1442 can be PCR amplification primers. One or more primers 1440 and 1442 can be sequencing primers. One or more adapters 1434 and 1436 can be used to further amplify adapter-tagged amplicon 1438. One or more adapters 1434 and 1436 can be used to sequence adapter-tagged amplicon 1438. Primer 1442 can include a plate index 1444 so that amplicons generated using the same barcode or random barcode set 1410 can be sequenced using next-generation sequencing (NGS) in one sequencing reaction.

[0216] Further details regarding library preparation workflows using systems of embodiments of the present invention (e.g., as described above) 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; and U.S. Patent Application Publication Nos. 2016 / 0340720; 2018 / 0276332; 2019 / 0338357; 2020 / 0124601; 2020 / 0157600; 2020 / 0255888; 2020 / 0299672; 2020 / 263169; 2020 / 040379, and 2021 / 0171940; the disclosures of which are incorporated herein by reference in their entirety.

[0217] sample

[0218] As described above, the method includes processing samples. 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 from a single cell. The sample for the disclosed method can include one or more cells. In some embodiments, a plurality of cells can include one or more cell types. At least one of the one or more cell types can be a brain cell, a heart cell, a cancer cell, a circulating tumor cell, an organ cell, an epithelial cell, a metastatic cell, a benign cell, a primary cell, a circulating cell, or any combination thereof. In some embodiments, the cell is a cancer cell removed from a cancerous tissue (e.g., breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, melanoma, and non-melanoma skin cancer, etc.). In some embodiments, the cell is derived from cancer, but is collected from a body fluid (e.g., circulating tumor cells). Non-limiting examples of cancer can include adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, and fibrosarcoma. The sample can include 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 a cell from a subject. The sample can be obtained from a human, a mammal, a dog, a rat, a mouse, a fish, a fly, a worm, a plant, a fungus, a bacterium, a virus, a vertebrate or an invertebrate.

[0219] In some embodiments, the cell is a cell that has been infected by a virus and contains a viral oligonucleotide. In some embodiments, the viral infection can be caused by a virus, such as a single-stranded (positive strand or "positive") DNA virus (e.g., a parvovirus) or a double-stranded RNA virus (e.g., a reovirus). In some embodiments, the cell is a bacterium. These bacteria can include gram-positive bacteria or gram-negative bacteria. In some embodiments, the cell is a fungus. In some embodiments, the cell is a protozoan or other parasite.

[0220] As used herein, the term "cell" may refer to one or more cells. In some embodiments, the cell is a normal cell, such as a human cell at a different stage of development, or a human cell from a different organ or tissue type. In some embodiments, the cell is a non-human cell, such as a mammalian cell of other types (e.g., mouse, rat, pig, dog, cow, and horse). In some embodiments, the cell is an animal or plant cell of other types. In other embodiments, the cell can be any prokaryotic cell or eukaryotic cell.

[0221] In some embodiments, before the cells are associated with the beads, the cells are sorted. For example, fluorescence activated cell sorting or magnetic activated cell sorting, or more commonly, flow cytometry can be used to sort the cells. The cells can be filtered by size. In some embodiments, the retentate comprises cells to be associated with the beads. In some embodiments, the flow-through comprises cells to be associated with the beads. The sample can refer to a plurality of cells. The sample can refer to a cell monolayer. The sample can refer to a thin section (e.g., a tissue thin section). The sample can refer to a solid or semi-solid cell collection that can be placed one-dimensionally on an array.

[0222] Reagent test kit

[0223] As described above, aspects of the present invention additionally include a test kit. The test kit of the present invention includes a multi-micropore array flow cell box of the present invention (e.g., as described above). In some cases, the test kit includes a plurality of multi-micropore array flow cell boxes. In some embodiments, the test kit includes a sample collection container holder configured to accommodate 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 test kit includes a plurality of sample collection containers. The test kit may also include one or more waste collection containers.

[0224] In some cases, the components of the subject test kit are provided in one or more sealable containers. In some cases, one or more sealable containers are resealable containers, i.e. they can be opened and closed. Any convenient container can be used, such as a pouch, bag, box, etc. In certain embodiments, a desiccant is provided in one or more containers, i.e., for controlling the moisture content in the container. Exemplary desiccant includes silica gel, etc. In some cases, the multi-micropore array flow cell box of the present invention can be used or partially used (e.g., by performing method of the present invention for one or more fluid swimming lanes of box), and is placed in a sealable container for storage. The multi-micropore array flow cell box used in part can be taken out from a sealable container, and for preparing one or more additional samples, after which, if necessary, the box can be put back in the container. The sealable container can be configured to maintain the stability of the partially used box so that it can continue to be used at one or more time points after the first use, such as 1 minute or more after the first use, such as 5 minutes or more after the first use, such as 30 minutes or more after the first use, such as 1 hour or more after the first use, such as 6 hours or more after the first use, such as 1 day or more after the first use, such as 1 week or more after the first use, such as 2 weeks after the first use, such as 1 month after the first use, such as 3 months after the first use, and including 6 months or more after the first use.

[0225] In some cases, the test kit includes barcoded beads, for example, wherein the barcoded beads include a nucleic acid barcode, which includes a universal primer binding domain, a cell marker 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 also includes a unique molecular index (UMI). In some embodiments, the test kit includes a random barcode that may not be attached to the beads. The test kit may also include reagents such as a lysis buffer (such as the buffer described above), a rinse / wash buffer, a hybridization buffer, and a reducing agent (such as dithiothreitol). In some embodiments, the test kit may also include reagents (such as enzymes, primers, dNTPs, NTPs, RNAse inhibitors, or buffers) for performing a nucleic acid extension reaction (such as a reverse transcription reaction and a primer extension reaction). In some embodiments, the test kit may also include reagents (such as enzymes, universal primers, sequencing primers, target-specific primers, or buffers) for performing an amplification reaction to prepare a sequencing library. In some embodiments, the test kit may include a ligase, a transposase, a reverse transcriptase, a DNA polymerase, an RNA enzyme, an exonuclease, or any combination thereof. In some embodiments, the test kit includes reagents for homopolymer tailing (e.g., terminal transferase and dNTP). The test kit may include reagents for, for example, any enzymatic cutting of the present disclosure (e.g., ExoI nuclease, restriction endonuclease). In some embodiments, the test kit may include reagents for adapter connection (e.g., ligase, reducing agent). In some embodiments, the test kit may include reagents for library preparation (e.g., adding sequencing library / flow cell primers), which may include sequencing / flow cell primers, enzymes for attaching primers, dNTPs, etc.

[0226] The components of the kit can be present in separate containers, or multiple components can be present in a single container. For example, the template-switching oligonucleotide and the template-switching polymerase can be present in the same tube or in different tubes. In certain embodiments, the components can be conveniently provided in a lyophilized form so that they are ready for use and can be conveniently stored at room temperature.

[0227] In addition to the above-mentioned components, the subject test kit can also include (in certain embodiments) the specification sheets for practicing the subject method.These specifications can be present in the subject test kit in various forms, and wherein one or more forms can be present in the test kit.A form of these specifications is the information printed on suitable medium or substrate, for example, one or more sheets of paper, test kit packaging, package inserts etc. of printed information thereon.Another form of these specifications is the computer-readable medium of record information thereon, for example, floppy disk, optical disc (CD), portable flash drive etc.Another form of these specifications that can exist is a website address, uses the website address to access the information of remote sites via the Internet.

[0228] For more detailed information on various aspects of the present invention, see 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; and U.S. Patent Application Publication Nos. 2016 / 0340720, 2018 / 0276332, 2019 / 0338357, 2020 / 0124601, 2020 / 0157600, 2020 / 0255888, 2020 / 0299672, 2020 / 263169, 2020 / 040379, and 2021 / 0171940; the disclosures of which are incorporated herein by reference in their entireties.

[0229] Practicality

[0230] System of the present invention, box, method and test kit can be used for preparing biological sample when needed, for example, for further analysis.For example, the present invention can be used for preparing biological sample for diagnosis, monitoring and research purpose.In some cases, system of the present invention, box, method and test kit can be used for improving sample preparation and the throughput of analysis, for example, improve 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, and comprise 8 times or more.In some cases, the present invention can be used for preparing and analyzing multiple samples simultaneously when needed, and can not produce batch effect.In some cases, system of the present invention, box, method and test kit can be used for simplifying and saving the mechanism of sample preparation.

[0231] The following examples are for illustrative purposes only and are not intended to be limiting.

[0232] experiment

[0233] Example 1

[0234] In COMSOL Simulations were performed in that demonstrate the magnetic forces generated by different magnet arrangements in a retrieval magnet assembly. Figure 15AShown is a magnetic force simulation of four 3" x 1 / 2" x 1 / 8" magnets with alternating polarity. Figure 15B Shown is a magnetic force simulation of four 3" x 1 / 2" x 1 / 8" magnets that do not have alternating polarity. Figure 15C A simulation of a 3"x3"x 1 / 8" magnet is shown. Figures 15A-15C As shown, magnets with alternating polarity exhibit a more uniform force.

[0235] A simulation was also performed to explore the effect of the distance between adjacent magnets on the magnetic force. Figure 15D Different magnet arrangements for the simulation are shown. Figures 15E-15G The results of the simulated arrangement are shown. Figure 15E A clear batch effect was observed in the arrangement shown.

[0236] against Figure 15D The different arrangements shown are compared parametrically. Figure 15I As shown, the "-2mm" and "aligned" magnet arrangements have similar magnetic field coverage on the centerline. Figure 15H As shown. Figure 15J As shown, the magnetic force is smallest along the center line.

[0237] Example 2

[0238] The number of cells captured per lane in an 8-lane cassette was measured. >40,000 cells / lane, or >320,000 viable cells ( Figure 16A Capture rates as high as 80% were observed. Multiplex rates were comparable to those of single-lane Express. The higher bead diversity resulted in a cell labeling collision rate of less than 0.5%. No batch effects were observed. A high correlation in gene expression was observed between multi-lane and single-lane Rhapsody ( Figure 16B ).

[0239] Example 3

[0240] Demonstrated the use of BDRhapsody TM HT-Xpress offers flexible cell throughput per lane, and results such as Figures 17A-17D All cell loadings were performed on a single 8-lane cassette, with 100, 1,000, 5,000, 10,000, 25,000, 40,000, 55,000, and 65,000 cells loaded simultaneously in lanes 1 through 8. The results correlated well with the theoretical Poisson multiplet loading values ​​for loadings of 10,000 or more cells and met the specifications for cell capture efficiency and multiplet rate.

[0241] Example 4

[0242] A single 8-lane cassette was partially used over multiple days. Some of lanes 1-8 were used at the same time, while the remaining lanes were used for the same or different assays at different times. 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 Figure 2. Figures 18A-18C shown.

[0243] Example 5

[0244] The stability of partially used cartridges was studied for up to 4 months. Figures 19A-19B Shown are the targeted +SMK assays performed on day 1 using 1:1 Jurkat / Ramos cells in lanes 1 and 2 of the 8-lane box. Figures 19C-19D Shown is a WTA+Abseq assay using PBMCs in lanes 3 and 4 of an 8-lane box. Days 1 and 7 are shown in separate lanes, showing close correlation to the single lane control.

[0245] Example 6

[0246] A 1:1:1 mix of cells of different sizes was run on an 8-lane cassette. Capture efficiency was similar for cells of different sizes (80% capture at 20K loading). No batch effects were observed for samples captured using either the single-lane or 8-lane cassettes. t-SNE analysis showed that Jurkat / BT549 / K562 cell clusters ( Figures 20A-20B ).

[0247] Example 7

[0248] The 8-lane cassette was evaluated for its ability to capture enriched neutrophils as well as sorted NK and T cells compared to a single-lane cassette. No batch effects were observed in samples captured using the single-lane and 8-lane cassettes. t-SNE analysis showed that the T cell / NK cell / neutrophil clusters ( Figure 21A ). Figure 21B The capture rates of different cells are shown.

[0249] Example 8

[0250] Prior to the Rhapsody workflow, 24 Jurkat / Ramos / THP1 cell sample tubes were stained with 24 Flex SMK tags and pooled. A targeted + SMK experiment was performed, showing >95% sensitivity and specificity for the 24 sample tags ( Figures 22A-22B A two-step staining with anti-β2 microglobulin as the primary antibody was used ( Figures 22C-22D ).

[0251] Example 9

[0252] Figure 23A A workflow is presented for characterizing full-length T cell receptor sequences as well as the transcriptome and surface proteins of antigen-specific T cells. Figure 23B Good resolution between Abseq (CITE-seq) and dCODE reads is shown. Figure 23C As shown, TCRα / β chain clonotypes were found in cell clusters with high antigen-specific CMV3 dCODE detection rates.

[0253] Example 10

[0254] The mRNA, surface proteins and intracellular proteins were profiled simultaneously. Figures 24A-24D High mRNA and surface Abseq correlation + / - intracellular Abseq is shown. Figures 24E-24L Intracellular CITE-seq detection of pSTAT1, pSTAT5, and pSTAT6 is shown. Intracellular CITE-seq results are consistent with flow cytometry results. Phosphorylation occurs in CD4 T cells after 15 minutes of stimulation with hIFN-α, hIL-2, and hIL-4.

[0255] Example 11

[0256] like Figures 25A-25C As shown, there is no batch effect between technical replicates between the two boxes containing labeled cells. The markers separated the samples into 6 different cell populations, and the results of the experiment show that the independence of the cell populations is preserved for each box and lane when superimposed on a t-SNE plot to illustrate the cell clusters of sample markers.

[0257] Notwithstanding the appended claims, the present disclosure is defined by the following clauses:

[0258] 1. A system comprising:

[0259] a tray configured to receive a multi-microwell array flow cell cartridge; and

[0260] A retrieval magnet assembly is configured to apply a uniform magnetic force to the flow cell of the cartridge when in an activated position.

[0261] 2. The system according to clause 1, wherein the retrieval magnet assembly is configured to apply the uniform magnetic force from a position above the tray.

[0262] 3. A system according to clause 1 or 2, wherein the uniform magnetic force is a magnetic field ranging from 650 Gauss to 1325 Gauss.

[0263] 4. The system according to any of the preceding clauses, wherein the retrieval magnet assembly is configured to apply the uniform magnetic force via a plurality of magnets.

[0264] 5. A system according to item 4, wherein the magnets in the plurality of magnets have alternating polarity.

[0265] 6. The system according to item 4 or 5, wherein the number of magnets in the retrieval magnet assembly ranges from 2 to 10.

[0266] 7. The system according to item 6, wherein the retrieval magnet assembly comprises 4 magnets.

[0267] 8. The system according to any one of items 4 to 7, wherein the plurality of magnetic elements comprise rare earth magnets.

[0268] 9. The system of clause 8, wherein the rare earth magnet is a neodymium magnet.

[0269] 10. The system of clause 8, wherein the rare earth magnet is a samarium cobalt magnet.

[0270] 11. The system according to any of the preceding clauses, wherein the magnets of the plurality of magnets are bar magnets.

[0271] 12. The system according to any of items 4 to 11, wherein the magnets of the plurality of magnets are ring magnets.

[0272] 13. The system of clause 12, wherein the magnets of the plurality of magnets are arranged in a bull's-eye configuration.

[0273] 14. A system according to any of the preceding items, wherein the retrieval magnet assembly is drivable between an activated position and an inactivated position, wherein the retrieval magnet assembly is located near the tray, and wherein the retrieval magnet is located farther from the tray relative to the activated position.

[0274] 15. The system according to any of the preceding items, further comprising a sample collection container holder configured to receive a plurality of sample collection containers for collecting analytes from the cartridge.

[0275] 16. The system according to item 15, further comprising said plurality of sample collection containers.

[0276] 17. The system according to item 16, wherein the system comprises a number of sample collection containers ranging from 2 to 10.

[0277] 18. The system according to item 16 or 17, wherein the system comprises a number of sample collection containers matching the number of flow cells in the cartridge.

[0278] 19. The system according to item 17 or 18, wherein the system comprises 8 sample collection containers.

[0279] 20. The system according to any one of items 15 to 19, wherein the specimen collection container holder comprises a counterweight configured to maintain the specimen collection container in an upright position.

[0280] 21. The system according to any one of items 15 to 20, wherein the system and the specimen collection container holder have complementary shapes such that the specimen collection container holder can be received in the system in a single orientation.

[0281] 22. The system of any of the preceding clauses, further comprising a drawer movable between a plurality of different positions within the system.

[0282] 23. The system according to any of the preceding items, further comprising a waste collection container for collecting liquid waste from the multi-well array flow cell cartridge.

[0283] 24. The system according to item 23, wherein the system comprises a single waste collection container.

[0284] 25. The system according to any of items 23 to 24, further comprising an interlock configured to prevent sample liquid from being collected into the waste collection container when the retrieval magnet assembly is in the activated position.

[0285] 26. The system according to any of the preceding items, wherein the tray comprises a latch for retaining the multi-microwell array flow cell cartridge.

[0286] 27. The system according to any of the preceding clauses, wherein the system does not comprise a lysis magnet positioned below the tray.

[0287] 28. The system according to any one of the preceding clauses, further comprising the multi-microwell array flow cell cartridge, wherein the multi-microwell array flow cell cartridge comprises:

[0288] Multiple fluid lanes, each containing:

[0289] an inlet for receiving a liquid;

[0290] a flow cell comprising a microwell array; and

[0291] Outlet for draining liquid.

[0292] 29. The system according to item 28, wherein the multi-microwell array flow cell cartridge comprises a number of fluid lanes ranging from 2 to 10.

[0293] 30. The system according to item 29, wherein the multi-microwell array flow cell cartridge comprises 8 fluid lanes.

[0294] 31. The system according to any one of items 28 to 30, wherein each outlet is stepped to prevent siphoning of liquid from the flow cell.

[0295] 32. The system according to any of items 28 to 31, wherein each flow cell comprises an elongated channel.

[0296] 33. The system according to item 32, wherein the length of the elongated channel ranges from 50 mm to 100 mm.

[0297] 34. The system according to any of items 28 to 33, wherein each microwell array comprises 250,000 microwells to 300,000 microwells.

[0298] 35. The system according to any one of items 28 to 34, wherein each microwell array comprises in the range of 36,000 microwells / cm 2 Up to 42,000 micropores / cm 2 density.

[0299] 36. The system according to any one of items 28 to 35, wherein the tray has a shape complementary to that of the multi-microwell array flow cell cartridge such that the multi-microwell array flow cell cartridge can be received in the tray in a single orientation.

[0300] 37. The system according to item 36, wherein the multi-microwell array flow cell cartridge comprises chamfered corners.

[0301] 38. The system according to any of the preceding clauses, further comprising a drip vessel positioned at a lower position of the tray, the drip vessel being configured to receive liquid drained from the cartridge.

[0302] 39. The system according to item 38, wherein the drip vessel is detachable.

[0303] 40. A method for processing a cell sample, the method comprising:

[0304] A multi-well array flow cell cartridge is introduced into the system, said system comprising:

[0305] a tray configured to receive the multi-microwell array flow cell cartridge; and

[0306] a retrieval magnet assembly configured to apply a uniform magnetic force to the flow cells of the multi-microwell array flow cell cartridge when in an activated position;

[0307] loading a sample into the multi-well array flow cell cartridge;

[0308] driving the retrieval magnet assembly to the activated position and applying the uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to generate a processed sample; and

[0309] The processed samples are collected from the multi-well array flow cell cartridge.

[0310] 41. The method according to item 40, wherein the method comprises loading a plurality of different samples into different flow cells of the cartridge.

[0311] 42. The method according to item 41, wherein the number of different samples ranges from 2 to 10.

[0312] 43. The method according to any one of items 40 to 42, further comprising loading a lysis buffer into the multi-well cartridge after loading the sample liquid into the multi-well cartridge.

[0313] 44. The method according to item 43, wherein the method comprises loading the lysis buffer into the multi-well cartridge without using a lysis magnet.

[0314] 45. Method according to any one of items 40 to 44, wherein said method comprises loading barcoded beads into said multi-well cartridge prior to said actuation of said retrieval magnet assembly.

[0315] 46. ​​The method according to item 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.

[0316] 47. The method according to item 46, wherein the target capture domain is a poly(T) sequence.

[0317] 48. The method according to item 46, wherein the nucleic acid barcode further comprises a unique molecular index (UMI).

[0318] 49. The method according to any of items 40 to 48, further comprising applying a uniform magnetic field from a position above the tray.

[0319] 50. The method according to any one of items 40 to 49, wherein the retrieval magnet assembly is configured to apply a uniform magnetic force to the flow cells of the multi-microwell array flow cell cartridge via a plurality of magnets when in the activated position.

[0320] 51. The method of clause 50, wherein the magnets in the plurality of magnets have alternating polarity.

[0321] 52. The method according to item 50 or 51, wherein the number of magnets in the retrieval magnet assembly ranges from 2 to 10.

[0322] 53. The method according to item 52, wherein the retrieval magnet assembly comprises 4 magnets.

[0323] 54. The method according to any one of items 50 to 53, wherein said plurality of magnetic materials comprises rare earth magnets.

[0324] 55. The method according to any of the preceding items 40 to 54, wherein said system comprises a sample collection container holder configured to receive a plurality of sample collection containers for collecting analytes from said sample in said cartridge.

[0325] 56. The method according to item 55, wherein the method comprises collecting the processed samples in a plurality of sample collection containers.

[0326] 57. The method according to item 56, wherein the method comprises collecting the processed samples in a number ranging from 2 to 10 sample collection containers.

[0327] 58. The method according to item 56 or 57, wherein the method comprises collecting the processed samples in a number of sample collection containers matching the number of flow cells in the cartridge.

[0328] 59. The method according to item 57 or 58, wherein the method comprises collecting 8 processed samples in 8 sample collection containers.

[0329] 60. The method according to any one of items 55 to 59, wherein the specimen collection container holder comprises a weight configured to maintain the specimen collection container in an upright position.

[0330] 61. The method according to any of the preceding items 40 to 60, wherein the system comprises a drawer movable between a plurality of different positions within the system.

[0331] 62. The method of clause 61, further comprising moving the drawer between the plurality of different positions within the system.

[0332] 63. The method according to any one of the preceding items 40 to 62, further comprising collecting waste liquid from the multi-microwell array flow cell cartridge in a waste collection container.

[0333] 64. The method according to any one of items 61 to 63, wherein the system comprises an interlock configured to prevent sample liquid from being collected into the waste collection container when the retrieval magnet assembly is in the activated position.

[0334] 65. The method of clause 62, further comprising engaging said interlock during said actuation of said retrieval magnet assembly.

[0335] 66. The method according to any one of items 40 to 65, wherein the multi-microwell array flow cell cartridge comprises:

[0336] Multiple fluid lanes, each containing:

[0337] an inlet for receiving a liquid;

[0338] a flow cell comprising a microwell array; and

[0339] Outlet for draining liquid.

[0340] 67. The method according to item 66, wherein the box comprises a number of fluid lanes ranging from 2 to 10.

[0341] 68. The method according to item 67, wherein the box comprises 8 fluid lanes.

[0342] 69. The method according to any of items 66 to 68, wherein the outlet is stepped to prevent siphoning of liquid from the flow cell.

[0343] 70. Method according to any of items 66 to 69, wherein each flow cell comprises an elongated channel.

[0344] 71. The method according to item 70, wherein the length of the elongated channel ranges from 50 mm to 100 mm.

[0345] 72. The method according to any one of items 66 to 71, wherein each microwell array comprises 250,000 microwells to 300,000 microwells.

[0346] 73. The method according to any one of items 66 to 72, wherein each microwell array comprises in the range of 36,000 microwells / cm 2 Up to 42,000 micropores / cm 2 density.

[0347] 74. The method according to any one of items 40 to 73, wherein the method further comprises generating a sequencing-ready nucleic acid library from the processed sample.

[0348] 75. The method according to item 74, wherein said sequencing-ready nucleic acid library is sequenceable by using a next-generation sequencing protocol.

[0349] 76. Method according to any one of items 40 to 75, wherein said method is a method of genomic analysis.

[0350] 77. Method according to any one of items 40 to 75, wherein said method is a method of epigenomic profiling.

[0351] 78. Method according to any one of items 40 to 75, wherein said method is a method of transcriptome profiling.

[0352] 79. Method according to any one of items 40 to 75, wherein said method is a method of proteomic analysis.

[0353] 80. Method according to any one of items 40 to 75, wherein said method is a method of multi-omics analysis.

[0354] 81. The method according to item 80, wherein said multi-omics analysis comprises at least transcriptomic analysis and proteomic analysis.

[0355] 82. A multi-microwell array flow cell cartridge comprising:

[0356] Multiple fluid lanes, each containing:

[0357] an inlet for receiving a liquid;

[0358] a flow cell comprising a microwell array; and

[0359] Outlet for draining liquid.

[0360] 83. The multi-microwell array flow cell cartridge according to item 82, wherein the multi-microwell array flow cell cartridge comprises a number of fluid lanes ranging from 2 to 10.

[0361] 84. The multi-microwell array flow cell cartridge according to item 83, wherein the multi-microwell array flow cell cartridge comprises 8 fluid lanes.

[0362] 85. The multi-microwell array flow cell cartridge according to any one of items 82 to 84, wherein the outlet is stepped to prevent siphoning of liquid from the flow cell.

[0363] 86. The multi-microwell array flow cell cartridge according to any one of items 82 to 85, wherein the outlet is a tapered orifice.

[0364] 87. The multi-microwell array flow cell cartridge according to item 86, wherein the tapered orifice is configured to achieve a channel flow rate of 20 μL / s to 500 μL / s to induce droplet formation.

[0365] 88. The multi-microwell array flow cell cartridge according to any one of items 82 to 87, wherein each flow cell comprises an elongated channel.

[0366] 89. The multi-microwell array flow cell cartridge according to item 88, wherein the length of the elongated channel ranges from 50 mm to 100 mm.

[0367] 90. The multi-microwell array flow cell cartridge according to any one of items 82 to 89, wherein each microwell array comprises 250,000 microwells to 300,000 microwells.

[0368] 91. The multi-microwell array flow cell cartridge according to any one of items 82 to 90, wherein each microwell array comprises in the range of 36,000 microwells / cm 2 Up to 42,000 micropores / cm 2 density.

[0369] 92. The multi-microwell array flow cell cartridge according to any one of items 82 to 91, wherein the multi-microwell array flow cell cartridge comprises chamfered corners.

[0370] 93. The multi-microwell array flow cell cartridge according to any one of items 82 to 92, wherein each inlet comprises a gasket.

[0371] 94. The multi-microwell array flow cell cartridge according to item 93, wherein the gasket comprises a Shore hardness ranging from 10 to 80.

[0372] 95. The multi-microwell array flow cell cartridge according to item 93, wherein the gasket is configured to conically lock onto a pipette tip.

[0373] 96. The multi-microwell array flow cell cartridge according to item 95, wherein the Z-axis tolerance of the taper lock is 0.5 mm.

[0374] 97. A sample container holder configured to receive a plurality of sample collection containers for collecting analytes from a multi-well array flow cell cartridge according to any one of items 82 to 96.

[0375] 98. The sample container holder according to item 97, wherein the sample container holder is configured to receive a number ranging from 2 to 10 sample collection containers.

[0376] 99. The sample container holder according to item 98, wherein the sample container holder is configured to receive 8 sample collection containers.

[0377] 100. The sample container holder according to any one of items 97 to 99, wherein the sample collection container holder comprises a counterweight configured to maintain the sample collection container in an upright position.

[0378] 101. The sample container holder according to any of items 97 to 100, wherein 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.

[0379] 102. A kit comprising:

[0380] A multi-microwell array flow cell cartridge, comprising:

[0381] Multiple fluid lanes, each containing:

[0382] an inlet for receiving a liquid;

[0383] a flow cell comprising a microwell array; and

[0384] Outlet for draining liquid.

[0385] 103. The kit according to item 102, wherein the box comprises a number of fluid lanes ranging from 2 to 10.

[0386] 104. The kit according to item 103, wherein the box comprises 8 fluid lanes.

[0387] 105. The kit according to any one of items 102 to 104, wherein each outlet is stepped to prevent siphoning of liquid from the flow cell.

[0388] 106. The kit according to any of items 102 to 105, wherein the outlet is a tapered orifice.

[0389] 107. The kit according to item 106, wherein the tapered orifice is configured to achieve a channel flow rate of 20 μL / s to 500 μL / s to induce droplet formation.

[0390] 108. The kit according to any of items 102 to 107, wherein each flow cell comprises an elongated channel.

[0391] 109. The kit according to item 108, wherein the length of the elongated channel ranges from 50 mm to 100 mm.

[0392] 110. The kit according to any of items 102 to 109, wherein each microwell array comprises 250,000 microwells to 300,000 microwells.

[0393] 111. The kit according to any one of items 102 to 110, wherein each microwell array comprises in the range of 36,000 microwells / cm 2 Up to 42,000 micropores / cm 2 density.

[0394] 112. The kit according to any one of items 102 to 111, wherein the multi-microwell array flow cell cartridge comprises chamfered corners.

[0395] 113. The kit according to any of items 102 to 112, wherein each inlet comprises a gasket.

[0396] 114. The kit according to item 113, wherein the gasket comprises a Shore hardness ranging from 10 to 80.

[0397] 115. The kit according to item 113, wherein the gasket is configured to conically lock onto a pipette tip.

[0398] 116. The kit according to item 113, wherein the Z-axis tolerance of the taper lock is 0.5 mm.

[0399] 117. The kit according to any one of items 102 to 116, wherein the kit comprises a plurality of multi-microwell array flow cell cartridges.

[0400] 118. The kit according to any one of items 102 to 117, further comprising a sample collection container holder configured to receive a plurality of sample collection containers.

[0401] 119. The kit according to item 118, wherein the sample collection container holder comprises a counterweight configured to maintain the sample collection container in an upright position.

[0402] 120. The kit according to any of items 102 to 119, further comprising a plurality of sample collection containers.

[0403] 121. The kit according to any of items 102 to 120, further comprising a waste collection container.

[0404] 122. The kit according to any of items 102 to 121, further comprising a cell lysis buffer.

[0405] 123. The kit according to any of items 102 to 122, further comprising a hybridization buffer.

[0406] 124. The kit according to any of items 102 to 123, further comprising a wash buffer.

[0407] 125. The kit according to any of items 102 to 124, further comprising a reducing agent.

[0408] 126. The kit according to any of items 102 to 125, further comprising barcoded beads.

[0409] 127. The kit according to item 126, wherein the barcoded beads comprise a nucleic acid barcode comprising a universal primer binding domain, a cell labeling domain and a target capture domain.

[0410] 128. The kit according to item 127, wherein the target capture domain is an oligo dT sequence.

[0411] 129. The kit according to item 127, wherein the nucleic acid barcode further comprises a unique molecular index (UMI).

[0412] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0413] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to think of various arrangements, although not explicitly described or shown herein, but these arrangements embody the principles of the present invention and are included in its spirit and scope. In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts that the inventors have contributed to promote this area, and should be interpreted as not being limited to the examples and conditions of such specific records. In addition, all statements recording the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover their structural and functional equivalents. In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, no matter how the structure, any element of the performance of the same function developed. In addition, anything disclosed herein is not intended to be dedicated to the public, regardless of whether such disclosure is clearly recorded in the claims.

[0414] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are 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 cited for a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" appears at the beginning of a limitation in the claim; if such exact phrase is not used in a limitation in a claim, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not cited.

Claims

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

4. A system according to any of the preceding claims, wherein the retrieval magnet assembly is actuatable between an activated position in which the retrieval magnet assembly is located near the tray and an inactivated position in which the retrieval magnet is located farther from the tray relative to the activated position.

5. The system according to any one of the preceding claims, 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 any preceding claim, further comprising a drawer movable between a plurality of different positions within the system.

7. The system of any one of the preceding claims, further comprising a waste collection container for collecting liquid waste from the multi-well array flow cell cartridge.

8. The system of any one of the preceding claims, wherein the tray comprises a latch for retaining the multi-microwell array flow cell cartridge.

9. The system of any preceding claim, wherein the system does not comprise a lysis magnet positioned below the tray.

10. The system according to any one of the preceding claims, further comprising the multi-microwell array flow cell cartridge, wherein the multi-microwell array flow cell cartridge comprises: Multiple fluid lanes, each containing: an inlet for receiving a liquid; a flow cell comprising a microwell array; and Outlet for draining liquid.

11. The system of any one of the preceding claims, further comprising a drip vessel positioned at a lower position of the tray, the drip vessel being configured to receive liquid drained from the cartridge.

12. A method for processing a cell sample, the method comprising: A multi-well array flow cell cartridge is introduced into the system, said system comprising: a tray configured to receive the multi-microwell array flow cell cartridge; as well as a retrieval magnet assembly configured to apply a uniform magnetic force to a flow cell in the multi-microwell array flow cell cartridge when in an activated position; loading a sample into the multi-well array flow cell cartridge; driving the retrieval magnet assembly to the activated position and applying the uniform magnetic force to the sample in the multi-microwell array flow cell cartridge to generate a processed sample; as well as The processed sample is collected from the multi-well array flow cell cartridge.

13. A multi-microwell array flow cell cartridge comprising: Multiple fluid lanes, each containing: an inlet for receiving a liquid; a flow cell comprising a microwell array; and Outlet for draining liquid.

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

15. A kit comprising: A multi-microwell array flow cell cartridge, comprising: Multiple fluid lanes, each containing: an inlet for receiving a liquid; a flow cell comprising a microwell array; and Outlet for draining liquid.

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