Reversible immobilization reagents and methods of use thereof

Abstract

The present disclosure relates to compositions and methods for reversible immobilization of biological samples using immobilization reagents that form bis-urethane crosslinks between amine-containing moieties in biomolecules.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 952,677, filed December 23, 2019, and U.S. Provisional Application No. 63 / 026,513, filed May 18, 2020, each of which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to fixation reagent compositions comprising bis-imidazole-carboxylate compounds and methods for using them to prepare reversibly fixed biological samples. Background Technology

[0004] Biological samples containing multiple biomolecules can be used for a variety of purposes, such as disease detection (e.g., cancer) and / or genotyping (e.g., species identification). Microfluidics have been developed to facilitate the processing of biological samples by dividing each sample (e.g., cells) into discrete partitions (e.g., pores or droplets) separate from other partitions (e.g., droplets fluidly isolated from other droplets). This division into discrete partitions allows for precise control of the individual environments interacting with the biological sample. Furthermore, biological samples divided into discrete partitions can be barcoded and subjected to chemical or physical processes, such as heating, cooling, or chemical reactions. Thus, each discrete partition can contain its own individual sample and be individually measured, either qualitatively or quantitatively.

[0005] However, biological samples are unstable. Physical degradation begins immediately upon removal from their viable ecological niche. The extent of degradation is determined by many factors, including time, solution buffering conditions, temperature, source (e.g., certain tissues and cells have higher levels of endogenous RNase activity), biological stress (e.g., enzymatic tissue dissociation can activate stress-response genes), and physical manipulation (e.g., pipetting, centrifugation). Degradation includes important nucleic acid molecules (e.g., RNA), proteins, as well as higher-order 3D structures of molecular complexes, intact cells, tissues, organs, and organisms. The instability of biological samples is a significant obstacle to their use in partition-based assays (e.g., droplet-based or well-based single-cell assays). Sample degradation greatly limits the ability to use such assays accurately and reproducibly across a wide range of available biological samples.

[0006] The instability of biological samples can be mitigated by using standard methods to fix samples, such as cryopreservation, dehydration (e.g., in methanol), high-salt storage (e.g., using RNAssist or RNAlater), and / or chemical fixatives that generate covalent crosslinks (e.g., paraformaldehyde or DSP). The ability to use such fixed biological samples in assays, particularly zonal assays, requires that the fixed biological samples be rapidly and efficiently defixed so that relevant assays can be performed before sample degradation occurs.

[0007] There remains a need for compositions and methods for reversibly immobilizing biological samples in a manner compatible with partition-based assays (e.g., microfluidic droplet-based or pore-based assays). Summary of the Invention

[0008] This disclosure provides compositions and methods that allow for reversible fixation of biological samples, and uses of these compositions and methods in partition-based assays (e.g., pore- or droplet-based analyses), such as gene expression profiling.

[0009] In at least one embodiment, this disclosure provides a method for preparing a biological sample, comprising contacting the sample with a first immobilization reagent composition containing a compound of formula (I).

[0010]

[0011] The "linker" comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12; optionally, m = 1. In at least one embodiment, the "linker" comprises: (a) a straight-chain alkane moiety of 2-24 carbons; (b) an ethylene glycol moiety; (c) a disulfide bond; and / or (d) a branched alkane moiety.

[0012] In at least one embodiment of the method, the compound of formula (I) is the compound of formula (II).

[0013]

[0014] Where n is between 1 and 13.

[0015] In at least one embodiment of the method, the compound of formula (I) is the compound of formula (III).

[0016]

[0017] Where n is between 1 and 12.

[0018] In at least one embodiment of the method, the compound of formula (I) is the compound of formula (IV).

[0019]

[0020]

[0021] Where m is from 1 to 13, and n is from 1 to 13.

[0022] In at least one embodiment of the method, the compound of formula (I) is the compound of formula (V).

[0023]

[0024] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0025] In at least one embodiment of the method, the compound of formula (I) is compound (6).

[0026]

[0027] In at least one embodiment of the method, the first fixative composition comprises: (a) a compound of formula (I) at a concentration of 50 mM or less, 25 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less; and / or (b) DMSO at a concentration of 5% or less, 2.5% or less, or 1.5% or less. In at least one embodiment of the method, the first fixative composition is contacted at room temperature (RT) for 3 hours or less, 60 minutes or less, 30 minutes or less, 15 minutes or less, or 5 minutes or less.

[0028] In at least one embodiment, the method further includes contacting the sample with a second fixation reagent composition; optionally, wherein the second fixation reagent composition comprises paraformaldehyde; optionally, wherein the paraformaldehyde concentration is 1% or lower.

[0029] In at least one embodiment of the method, the first immobilizing agent composition comprises a compound of formula (II).

[0030]

[0031] Wherein, n is 1 to 13; the second immobilization reagent composition comprises a compound of formula (III):

[0032]

[0033] Where n is between 1 and 12.

[0034] In at least one embodiment of the method, the first fixative composition comprises a compound of formula (III), wherein n is 1 to 12; and the second fixative composition comprises a compound of formula (II), wherein n is 1 to 13.

[0035] In at least one embodiment of the method, the method further includes contacting the sample with a de-fixing agent; optionally, the de-fixing agent comprises: (a) a compound capable of cleaving urethane bonds; optionally, said compound capable of cleaving urethane bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof; (b) a compound capable of cleaving disulfide bonds; optionally, said compound capable of cleaving disulfide bonds is DTT; and / or (c) a compound capable of reversing paraformaldehyde fixation.

[0036] In at least one embodiment, this disclosure also provides a composition comprising a fixed biological sample, wherein the sample comprises a cross-linked biomolecule of formula (Ia).

[0037]

[0038] Among them, X 1 and X 2 The "linker" is the amine-containing portion of the same or different biomolecules in the sample. The "linker" comprises an ethylene glycol portion and / or a straight-chain or branched-chain alkane portion of 2-24 carbons; m is 1 to 12. In at least one embodiment, the "linker" comprises: (a) a straight-chain alkane portion of 2-24 carbons; (b) an ethylene glycol portion; (c) a disulfide bond; and / or (d) a branched-chain alkane portion.

[0039] In at least one embodiment of the composition, the immobilized biological sample contains cross-linked biomolecules of formula (IIa).

[0040]

[0041] Where n is between 1 and 13.

[0042] In at least one embodiment of the composition, the immobilized biological sample contains cross-linked biomolecules of formula (IIIa).

[0043]

[0044] Where n is between 1 and 12.

[0045] In at least one embodiment of the composition, a cross-linked biomolecule of the immobilized biological sample inclusion formula (IVa) is included.

[0046]

[0047] Where m is from 1 to 13, and n is from 1 to 13.

[0048] In at least one embodiment of the composition, a cross-linked biomolecule of the immobilized biological sample inclusion formula (Va) is included.

[0049]

[0050] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0051] In at least one embodiment of the composition, a cross-linked biomolecule of the immobilized biological sample inclusion formula (VIa) is included.

[0052]

[0053] In at least one embodiment of the composition, the fixed biological sample is derived from a tissue sample, a biopsy sample, or a blood sample; optionally, the fixed biological sample is a single cell.

[0054] In at least one embodiment of the composition, the fixed biological sample has been fixed by a fixation reagent composition comprising formula (I), (II), (III), (IV), (V) or compound (6).

[0055] In at least one embodiment of the composition, the immobilized biological sample is provided together with a lysing agent and / or a de-solidifying agent in discrete partitions (e.g., provided or encapsulated in discrete droplets or provided in pores); optionally, the de-solidifying agent comprises a compound capable of cleaving urethane bonds; optionally, the compound capable of cleaving urethane bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof.

[0056] In at least one embodiment of the composition, the immobilized biological sample is provided in discrete partitions (e.g., provided or encapsulated in discrete droplets or provided in pores), wherein the discrete partitions further comprise a scaffold (e.g., beads): optionally, wherein the disintegrating agent is contained in the scaffold (e.g., beads).

[0057] In at least one embodiment of the composition, the immobilized biological sample is provided in discrete partitions (e.g., provided or encapsulated in discrete droplets or provided in discrete pores), wherein the discrete partitions also contain assay reagents; optionally, wherein the assay reagents are provided as part of a scaffold (e.g., beads) or contained within a scaffold (e.g., beads).

[0058] In at least one embodiment of the composition, the immobilized biological sample is provided in discrete partitions (e.g., provided or encapsulated in discrete droplets or provided in discrete pores), wherein the discrete partitions optionally also contain barcodes, wherein the barcodes are included as part of a scaffold (e.g., beads).

[0059] In at least one embodiment, this disclosure also provides a assay method comprising: (a) generating discrete partitions containing a fixed biological sample (e.g., droplets containing or encapsulating the fixed biological sample), a lysing agent and / or a deconsolidating agent, and an assay reagent, wherein the fixed sample contains a crosslink of formula (Ia).

[0060]

[0061] Among them, X 1 and X 2 The amine-containing moiety is the same or different biomolecule in the sample. The "linker" comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12; the decontaminating agent comprises a compound capable of cleaving carbamate bonds; optionally, said compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase or a combination thereof; (b) analytes that detect the reaction from the assay reagent and the decontaminating biological sample.

[0062] In at least one embodiment, this disclosure also provides a kit comprising: an assay reagent; and a fixation reagent composition containing a compound of formula (I).

[0063]

[0064] The "linker" comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12. In at least one embodiment, the "linker" comprises: (a) a straight-chain alkane moiety of 2-24 carbons; (b) an ethylene glycol moiety; (c) a disulfide bond; and / or (d) a branched alkane moiety. In at least one embodiment, the immobilizing agent composition comprises a compound of any one of formulas (II), (III), (IV), (V), or compound (6). Attached Figure Description

[0065] A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as “Figures” and “Diagrams”) illustrating illustrative embodiments (in which the principles of this disclosure are utilized), wherein:

[0066] Figure 1 An embodiment of a microfluidic channel structure for partitioning individual biological particles is shown.

[0067] Figure 2 An embodiment of a microfluidic channel structure for delivering beads carrying barcodes to droplets is shown.

[0068] Figure 3 An example of a microfluidic channel structure for co-dividing biological particles and reagents is shown.

[0069] Figure 4 An embodiment of a microfluidic channel structure for controlled partitioning of beads into discrete droplets is shown.

[0070] Figure 5 An embodiment of a microfluidic channel structure for increasing droplet generation flux is shown.

[0071] Figure 6 Another embodiment of a microfluidic channel structure for increasing droplet generation flux is shown.

[0072] Figure 7 Cell counts of different PBMC cell types found in fresh cells are described, compared with cells fixed with compound (2a) after 3 days of storage followed by defixation with CES and hydrazine, as described in Example 7.

[0073] Figure 8 An exemplary bead carrying a barcode is shown.

[0074] Figure 9 Another example of a bead carrying a barcode is shown.

[0075] Figure 10 A schematic diagram of an exemplary microporous array is shown.

[0076] Figure 11 An exemplary microporous array workflow for processing nucleic acid molecules is shown.

[0077] Figure 12 An example of the marker is illustrated schematically.

[0078] Figure 13 An example of beads carrying barcodes is described.

[0079] Figure 14A , 14B The workflow of an example of processing nucleic acid molecules is illustrated in 14C. Detailed Implementation

[0080] In the description herein and in the appended claims, unless the context clearly indicates otherwise, the singular form “a / an” includes multiple indicators. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prior basis for the use of exclusive terms such as “solely”, “only”, or the use of “negative” limitations in the description of claim elements. “comprise / comprises / comprising” and “include / includes / including” are used interchangeably and are not intended to be restrictive. It should also be understood that where the term “comprises” is used in the description of various embodiments, those skilled in the art will understand that in some specific instances, the language “consistently of…” or “composed of…” may be used alternatively to describe the embodiments.

[0081] If a range of values ​​is provided, unless the context explicitly specifies otherwise, it should be understood that each intermediate integer of the value and each tenth of each intermediate integer of the value, unless the context explicitly specifies otherwise, between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range, are included in embodiments of this disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also included in embodiments of this disclosure, subject to any specific exclusions of the range. Where the range includes one or both of these limitations, the range excluding (i) one or (ii) both included limitations is also included in embodiments of this disclosure. For example, “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”, etc.

[0082] Generally, the terminology used herein, as well as the techniques and procedures described herein, include those that are well understood and commonly used by one of ordinary skill in the art, such as those described by Sambrook et al. in Molecular Cloning: A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, New York, 1989 (hereinafter “Sambrook”); and in Current Protocols in Molecular Biology, edited by FMAusubel et al., Current Protocols, a joint venture of Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2011 Supplement) (hereinafter “Ausubel”).

[0083] All publications, patents, patent applications and other documents cited in this disclosure are incorporated herein by reference in their entirety for all purposes, to the same extent that each individual publication, patent, patent application or other document was individually indicated to be incorporated herein by reference.

[0084] 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 disclosure pertains. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. For the purposes of interpreting this disclosure, the following description of the terminology will apply, and where appropriate, terms used in the singular will also include plural forms, and vice versa.

[0085] A. Immobilization reagent composition of bis-imidazolium-carboxylate compounds

[0086] This disclosure provides compositions and methods that allow for the reversible fixation of biological samples. The ability to reversibly fix biological samples is based on fixation reagent compositions using a bis-imidazolium-carboxylate compound comprising general formula (I):

[0087]

[0088] The “linker” schematically described in Formula (I) can be any chemical group capable of covalently linking the imidazole carboxylic acid ester moiety. In at least one embodiment of the compound of Formula (I), the “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12.

[0089] The compound of formula (I) can be prepared by reacting carbonyl diimidazole (CDI) with the desired dihydroxy linker compound, as described in scheme 1.

[0090] Option 1

[0091]

[0092] Typical CDI synthesis reaction conditions can be used, such as 1.5 to 4 times excess CDI in dichloromethane, for 16 hours at room temperature, as described in Example 1. The general CDI-based reaction of Scheme 1 can be used with a variety of dihydroxy linker compounds (e.g., glycerol, dihydroxyalkanes, PEG compounds, hydroxylated branched alkyl compounds) to provide various fixed reagent compounds of formula (I).

[0093] Therefore, in at least one embodiment, this disclosure also provides a fixation reagent composition, wherein the compound of formula (I) is selected from: (a) compounds of formula (II)

[0094]

[0095] Where n is from 1 to 13;

[0096] (b) Compound of formula (III)

[0097]

[0098] Where n is from 1 to 12;

[0099] (c) Compound (IV)

[0100]

[0101] Where m is from 1 to 13, and n is from 1 to 13;

[0102] (d) Compound (V)

[0103]

[0104] Where R is -H, -O-(CO)-CH3, or -O-(CO)-imidazole

[0105] or

[0106] (e) Compound (6)

[0107]

[0108] Consider additional specific bis-imidazolium-carboxylate compounds of formulas (I), (II), (III), (IV), and (V). Table 1 provides a non-exhaustive list of such compounds used as immobilizing agents in the compositions and methods of this disclosure.

[0109] Table 1

[0110]

[0111]

[0112]

[0113]

[0114] B. Preparation of biological samples using bis-imidazolium-carboxylate compounds

[0115] The bis-imidazolium-carboxylate compounds of formula (I) (and subgenus compounds of formulas (II), (III), and (IV)), (V) and Table 1) provide advantageous technical effects for rapid and efficient reaction with the amine-containing moieties of biomolecules in biological samples (e.g., lysine and arginine side chains of proteins; adenine and guanine bases of nucleic acids) to form biscarbamate crosslinks. A generalized biological sample fixation reaction using the fixation reagent of formula (I) is shown in Scheme 2.

[0116] Option 2

[0117]

[0118] As used herein, the term "biosample" refers to any biologically derived sample that includes biomolecules. The term "biomolecule" is intended to encompass proteins, peptides, nucleic acids, carbohydrates, lipids, and all other biological macromolecules. Biosamples used in the methods and compositions of this disclosure include liquid samples of blood and other biological sources, solid tissue samples such as tissue samples (i.e., tissue specimens), biopsies (i.e., biopsy specimens), or tissue cultures or cells derived therefrom and their progeny. This includes samples that have been manipulated in any way after separation from a biological source, such as by treatment with reagents (e.g., fixation reagents, thereby generating fixed biosamples); samples, such as tissue embedded in a medium (e.g., paraffin); sectioned tissue samples (e.g., sectioned samples mounted on a solid substrate, such as glass slides); washed; or enriched with certain cell populations, such as cancer cells, neurons, stem cells, etc. The term also includes samples enriched with specific types of molecules, such as nucleic acids, peptides, etc. Biosamples also include clinical samples, including tissue obtained by surgical resection, tissue obtained by biopsy, cultured cells, cell supernatants, cell lysates, tissue samples (i.e., tissue specimens), organs, bone marrow, blood, plasma, serum, etc. Biological samples may also include samples obtained from the patient’s cancer cells, such as samples containing polynucleotides and / or peptides obtained from the patient’s cancer cells (e.g., cell lysates or other cell extracts containing polynucleotides and / or peptides); and samples having cells (e.g., cancer cells) from the patient.

[0119] As used herein, the term "fixation" for biological samples refers to a state of preservation against decay and / or degradation. "Fixation" also refers to the process of producing a fixed sample, which may include contacting biomolecules within the biological sample with a fixative agent (or "fixing agent") for a period of time, wherein the fixative agent reacts with the biomolecules of the sample to form covalent crosslinks between the chemical portions of the biomolecules (e.g., amine-containing portions). The reaction of the bis-imidazolium-carboxylate fixative agent with the amine-containing portion of the biomolecules is described in more detail below.

[0120] In this document, "unfixation" refers to the treatment state of cells, multiple cells, tissue samples, or any other biological samples, characterized by a previous fixed state followed by a reversal of the previous fixed state. For example, unfixed cells may also be referred to as "previously fixed" cells. In one embodiment, unfixed cells are characterized by the breaking or reversal of covalent bonds in the biomolecules of the cells or samples, wherein such covalent bonds were previously formed by treatment with the fixatives described herein.

[0121] The general biological sample fixation reaction of Scheme 2 can be carried out using a fixation reagent composition containing compound (I) under standard aqueous conditions typically used for biological sample fixation. The time for contacting the biological sample with the fixative to provide fixed biological samples depends on the temperature, sample properties, and the fixative used. For example, as described elsewhere herein and in examples, isolated cell samples are incubated with a small amount of a 5-15 mM solution of compound (2a) at room temperature for 30-60 minutes before quenching with excess buffer. The resulting fixed biological samples can be stored for days, weeks, or months under typical cryopreservation conditions before use.

[0122] Therefore, in at least one embodiment, this disclosure provides a method for preparing a biological sample, the method comprising contacting the sample with a first immobilization reagent composition containing a compound of formula (I).

[0123]

[0124] The “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12. It is further envisioned that the method can be implemented in which the compound of formula (I) is any one of the compounds of formula (II), (III), (IV) or (V), or compound (6), or any one or more bis-imidazolium-carboxylate compounds disclosed in Table 1.

[0125] For example, the bioimmobilization reaction of this method can be carried out using the immobilization reagent compound of formula (IV), as described in scheme 3.

[0126] Option 3

[0127]

[0128] Similarly, bioimmobilization reactions can be carried out using immobilization reagent compounds of formula (I), where m = 3. Such reactions, which can be carried out using reagents such as compound (6), result in branched dicarboxylate crosslinking between biomolecules in the biological sample, as shown in the generalized reaction of scheme 4.

[0129] Option 4

[0130]

[0131] C. Method using a fixed reagent combination

[0132] It is also envisioned that mixtures of bis-imidazolium carboxylate fixation reagent compounds of formula (I) can be used in the methods of this disclosure. For example, in at least one embodiment, the fixation reagent composition comprises a compound of formula (II) containing a linker having an alkyl chain, and a compound of formula (III) containing a linker having an ethylene glycol chain. Without being bound by theory, it is envisioned that the compound of formula (III), having an ethylene glycol chain “linker” moiety, is more readily soluble in aqueous solutions and is well-suited for immobilizing biomolecules on cell surfaces. In contrast, it is envisioned that the fixation reagent compound of formula (II) is less soluble in aqueous environments and is better suited for immobilizing intracellular biomolecules, having a more hydrophobic alkyl chain in its linker moiety. Thus, in at least one embodiment, the method can be performed, wherein the method comprises contacting a sample with a first fixation reagent composition containing compounds of formula (II) and (III). Generally, the selection of a particular bis-imidazolium carboxylate compound used in a fixation reagent composition useful in methods for preparing biological samples is guided by factors typically considered in the development of sample preparation methods. These factors include, but are not limited to, reagent solubility and compatibility with the type of biological sample, the conditions of use, and the required fixation / preservation volume.

[0133] Furthermore, the bis-imidazolium carboxylate fixative compound of formula (I) can be used in combination with a second fixative compound that does not form a bis-carboxylate crosslink. Examples of fixative compounds that can be used in combination with the bis-imidazolium carboxylate fixative compound of formula (I) include, but are not limited to, aldehyde fixatives (e.g., formaldehyde, commonly also referred to as "paraformaldehyde" and "formalin"; glutaraldehyde, etc.), imine esters, NHS (N-hydroxysuccinimide) esters, etc.

[0134] In some embodiments, the second fixative useful in the methods of this disclosure is paraformaldehyde (or "PFA"). Generally, the term "paraformaldehyde" in fixative is used interchangeably with "formaldehyde" and "formalin." Therefore, biological samples fixed with PFA may also be referred to as formalin fixation or formaldehyde fixation. Protocols and methods for using PFA as a biological sample fixative are well known in the art and can be used in the methods and compositions of this disclosure.

[0135] PFA is a robust fixative that crosslinks the bases of nucleic acids in a sample with amine bonds that are difficult to reverse (or deactivate) under conditions that maintain the integrity of the nucleic acids for further assays (e.g., in RNA analysis). Typically, a 4% PFA fixative solution is used to prepare biological samples. It is anticipated that significantly lower concentrations of PFA can be used in methods for preparing biological samples when combined with bis-imidazolium carboxylate fixative compounds of formula (I). For example, a biological sample can be treated with a first fixative composition containing compound (2a) followed by a second fixative composition containing 1% PFA. Without intending to be bound by theory, it is believed that the initial treatment with the bis-imidazolium carboxylate fixative of compound (2a) results in the fixation of mRNA with reversible DETA biscarbamate crosslinks, and the secondary treatment with 1% PFA stabilizes proteins and other biomolecules with amino crosslinks. However, given the prior treatment with compound (2a) for a period of time (e.g., >7 days), using only 1% PFA provides sufficient sample stability and also allows for adequate fixation and assays, such as RNA expression profiling. Therefore, in at least one embodiment, this disclosure provides a method for preparing a biological sample, including contacting the sample with a first fixation reagent composition comprising a compound of formula (I) and a second fixation reagent composition, wherein the second fixation reagent composition comprises PFA at a concentration of 2% or less, 1% or less, or 0.5% or less.

[0136] Conditions for reversing the PFA fixation effect on biological samples are known in the art; however, these conditions are often harsh. See, for example, WO2001 / 46402; US2005 / 0014203A1, and US2009 / 0202998A1. For example, the treatment of PFA-treated tissue samples involves heating in Tris buffer to 60–70 °C for several hours, but typically only removes a portion of the PFA-induced crosslinks. Using such harsh deconsolidation conditions can cause permanent damage to biomolecules in the sample, particularly nucleic acids. For example, Karmakar et al., “Organocatalytic removal of formaldehyde adducts from RNA and DNA bases,” *Nature Chemistry*, 7:752–758 (2015), describe a less harsh technique and conditions using deconsolidation compounds capable of catalytically cleaving amine bonds generated by PFA fixation; US 2017 / 0283860A1; US ​​2019 / 0135774A1. See, for example, Karmakar et al., “Organocatalytic removal of formaldehyde adducts from RNA and DNA bases,” *Nature Chemistry*, 7:752-758 (2015); US2017 / 0283860A1; and US2019 / 0135774A1. Therefore, in at least one embodiment of the method disclosed herein, it is contemplated that the method may further include the use of a catalytic destabilizing compound capable of reversing PFA fixation. Exemplary catalytic compounds capable of reversing PFA fixation are disclosed in Table 2 below.

[0137] Table 2

[0138]

[0139]

[0140]

[0141]

[0142] Compounds (7a)-(7f), (7l) and (7n) are commercially available. Compounds (7g), (7h), (7i), (7j), (7k), (7m) and (7o) can be prepared from commercially available reagents using standard chemical synthesis techniques well known in the art. See, for example, Crisalli et al., “The Importance of Ortho-Proton Donors in Hydrazone Formation Catalysis,” Organic Chemistry Communications, 2013, 15, 7, 1646-1649.

[0143] In short, compound (7h) can be prepared from a commercially available compound via a two-step synthesis as follows. Step 1: Diethyl(4-aminopyridin-3-yl)phosphonate was prepared according to the procedure described in Guilard, R. et al., Synthesis, 2008, 10, 1575-1579. In short, diethyl phosphite (2.2 mL, 17.3 mmol, 1.2 equiv), triethylamine (3 mL, 1.5 equiv), PPh3 (1.1 g, 4.3 mmol, 30 mol%), and Pd(OAc)2 (0.39 g, 1.73 mmol, 12 mol%) were added to a solution of 3-bromopyridin-4-amine (2.5 g, 14.5 mmol, 1 equiv) in ethanol (58 mL). The reaction mixture was purged with argon for 5 min. After heating to reflux for 24 hours, the reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was purified by silica gel chromatography (MeOH / DCM) to give diethyl (4-aminopyridin-3-yl)phosphate. 1 ¹H NMR (80 MHz, CDCl₃): δ = 1.15 (t, 6H, CH₃), 4.18–3.69 (m, 4H, CH₂), 5.99 (br-s, 2H, NH₂), 6.49 (d, 1H), 8.03–7.93 (m, 1H), 8.22 (d, 1H)). Step 2: The precursor compound (4-aminopyridin-3-yl) phosphate diethyl ester (0.35 g, 1.52 mmol, 1 equiv) from Step 1 was suspended in 6N HCl (aqueous solution) (8 mL). After reflux for 12 hours, the reaction mixture was concentrated under vacuum. The residue was washed with DCM and diethyl ether and concentrated under vacuum to give the target 4-aminopyridin-3-ylphosphonic acid of the compound (7 h). 1 H NMR (80MHz, D2O): δ=6.85-6.55(m, 1H), 8.05-7.94(m, 1H), 8.40-8.26(m, 1H)0.

[0144] Compound (7k) can be prepared by a four-step synthesis from the following commercially available compound. Step 1: Triethyl phosphite (1.9 mL, 10.8 mmol, 1.5 equiv) and palladium (OAc)₂ (0.16 g, 0.72 mmol, 10 mol%) were added to a solution of methyl 4-amino-3-iodobenzoate (2 g, 7.2 mmol, 1 equiv) in acetonitrile (20 mL). The reaction mixture was purged with argon for 5 min. After heating to reflux for 18 hours, the reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was partitioned between ethyl acetate and water, and the organic layer was dried with MgSO₄ and concentrated under vacuum. The crude mixture was purified by silica gel chromatography (ethyl acetate / hexane) to give methyl-4-amino-3-(diethoxyphosphoryl)benzoate ( 1 ¹H NMR (500 MHz, DMSO-d6): δ = 1.27 (t, 6H, CH₃), 3.80 (s, 3H, OMe), 3.97–4.11 (m, 4H, OCH₂), 6.76 (br-s, 2H, NH₂), 6.80–6.83 (m, 1H), 7.82 (dd, 1H), 7.98 (dd, 1H)). Step 2: To a solution of methyl-4-amino-3-(diethoxyphosphoryl)benzoic acid (0.96 g, 3.15 mmol, 1 equiv) in tetrahydrofuran: methanol:water (10 mL: 2.5 mL, ratio: 4:1:1), solid LiOH (0.45 g, 18.9 mmol, 6 equiv) was added. After heating at 60 °C for 6 hours, the reaction mixture was concentrated under vacuum, acidified to pH 2, and a solid precipitated. The solid was filtered and washed twice with 1N HCl to give 4-amino-3-(diethoxyphosphoryl)benzoic acid (0.49 mg, 57% yield). 1¹H NMR (80 MHz, CDCl₃): δ = 1.34 (t, 6H, CH₃), 3.85–4.38 (m, 4H, OCH₂), 5.74 (br-s, 2H, NH₂), 6.50–6.76 (m, 1H), 7.86–8.36 (m, 2H)). Step 3: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM) was added to a solution of 4-amino-3-(diethoxyphosphoryl)benzoic acid (0.25 g, 0.92 mmol, 1 equiv) and polyethylene glycolamine (0.75 g, 1.01 mmol, 1.1 equiv) in methanol (4.6 mL) under argon atmosphere. After stirring for 18 h at room temperature, the reaction mixture was concentrated under vacuum, and the residue was partitioned between DCM and brine. The organic layer was washed with 1N HCl and saturated sodium bicarbonate solution, dried with MgSO4, filtered, and concentrated under vacuum to obtain polyethylene glycol amide ethyl phosphate (0.35 g, 36% yield), which was then proceeded to the next step without purification. 1 ¹H NMR (80 MHz, CD₃OD): δ = 1.34 (t, 6H, CH₃), 3.44 (s, 3H, OCH₃), 3.56–3.91 (m, PEG), 4.02–4.22 (m, 4H, OCH₂), 6.62–6.93 (m, 1H), 7.74–8.45 (m, 2H). Step 4: The polyethylene glycol amide ethyl phosphonate from Step 3 (0.35 g, 0.36 mmol, 1 equiv) was suspended in 6N HCl (aqueous solution) (8 mL). After reflux for 12 hours, the reaction mixture was concentrated under vacuum. The residue was washed with methanol and DCM and concentrated under vacuum to give the compound (7k) as PEG amide phosphonate (0.31 g, 94% yield). 1 H NMR (80MHz, D2O): δ=3.02-4.06 (m, PEG), 7.36-7.52 (m, 1H), 7.99-8.09 (m, 1H)).

[0145] Compounds (7i) and (7j) can be prepared by a similarly simple procedure. For example, compound (7i) can be prepared in two steps from 2-bromopyridin-3-amine (CAS No. 39856-58-1; Sigma-Aldrich, St. Louis, Missouri), as shown in the figure below.

[0146]

[0147] Compound (7j) was prepared from 4-bromopyrimidine-5-amine (CAS No. 849353-34-0; Ambid Corporation, Arlington Heights, Illinois, USA) in a similar two-step manner, as shown in the figure below.

[0148]

[0149] Proline analogs (7m) and (7o) are prepared from commercially available protected precursor compounds via direct, one-step deprotection. Therefore, in at least one embodiment of the method of this disclosure, which includes the use of PFA, the method may further include contacting the sample with a destabilizing agent capable of reversing PFA fixation, optionally wherein the destabilizing agent is selected from any of the compounds (7a)-(7o) as shown in Table 2.

[0150] D. Compositions of dicarboxylate crosslinked biomolecules

[0151] As described in Schemes 2, 3, and 4, the use of a diimidazolium carboxylate compound of formula (I) in the method for immobilizing biological samples results in the formation of dicarbamate crosslinks between the amine-containing moieties (e.g., proteins and nucleic acids) of the sample's biomolecules. The dicarbamate crosslinks formed between the amine-containing moieties of the biomolecules due to treatment with the compound of formula (I) can be represented by the structure of formula (Ia).

[0152]

[0153] The structure of formula (Ia) describes the amine-containing portion of a biomolecule as X 1 and X 2 And we can imagine X 1 and X 2 It can be a part of the same or different biomolecules of a biological sample. The “linker” can vary as in the compound of formula (I) and includes an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12.

[0154] The bis(Ia) carbamate crosslinked biomolecule represents the “fixed” component of a fixed biological sample. As with other known fixation methods, the ability of covalent bis(Ia) crosslinking to reduce and / or inhibit the chemical processes that lead to degradation of the biological sample is believed. Therefore, this disclosure also provides fixed biomolecules comprising a covalent bis(Ia) carbamate structure not found in natural biomolecules. In at least one embodiment, this disclosure provides a composition comprising a fixed biological sample, wherein the sample comprises a crosslink of formula (Ia), wherein X 1 and X 2 "Amine" refers to the amine-containing portion of the same or different biomolecules in the sample; "linker" includes the ethylene glycol portion and / or the straight-chain or branched-chain alkane portion of 2-24 carbons; m is 1 to 12.

[0155] Furthermore, in at least one embodiment, this disclosure also provides a composition comprising a fixed biological sample, wherein the sample comprises: (a) a cross-linked biomolecule of formula (IIa).

[0156]

[0157] Where n is from 1 to 13;

[0158] (b) Cross-linked biomolecules of formula (IIIa)

[0159]

[0160] Where n is from 1 to 12;

[0161] (c) Cross-linked biomolecules of formula (IVa)

[0162]

[0163] Where m is from 1 to 13, and n is from 1 to 13;

[0164] (d) Cross-linked biomolecules of formula (Va)

[0165]

[0166] Wherein R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole; and / or

[0167] (e) Cross-linked biomolecules of formula (VIa)

[0168]

[0169] E. Instructions for using the desiccant

[0170] As shown in Scheme 2 above, the immobilized biosample prepared using the immobilization reagent of this disclosure (e.g., compound of formula (I)) contains dicarbamate crosslinks. The dicarbamate crosslinks used to immobilize biomolecules within biomolecules having the structure of formula (Ia) can be reversed using standard carbamate cleavage compositions and methods. Therefore, another advantageous technical effect of the immobilization reagent compositions and methods of this disclosure is their ease of reversibility. The generalized decoupling reaction reversing dicarbamate crosslinks is as follows: Figure 5 As shown,

[0171] Option 5

[0172]

[0173] Therefore, the method for preparing biological samples using the imidazole carboxylate fixation reagent compound of formula (I) can further include a subsequent step of reversing fixation by contacting the sample with a de-fixing agent. As shown in Scheme 5, the compound diethylenetriamine (“DETA”) is effective for cleaving carbamate bonds. See, for example, Noshita et al., “Direct Cleavage of Unactivated Carbamates and Urea Mediated by Diethylenetriamine,” Organic Chemistry Communications, 18: 6062-6065 (2016). Similarly, diamine compounds, including ethylenediamine (“EDA”), triethylenetetramine (“TETA”), and hydrazine monohydrate, can cleave carbamate bonds. Carboxylesterases are capable of cleaving carbamate bonds, and it is anticipated that carboxylesterases can also be used as de-fixing agents in the methods of this disclosure. Therefore, in at least one embodiment, this disclosure provides a method for preparing biological samples by contacting a sample with a fixation reagent composition comprising a compound of formula (I), wherein the method further includes contacting the sample with a de-fixing agent, wherein the de-fixing agent is a compound capable of cleaving carbamate bonds. In at least one embodiment including a decongestant, the compound capable of cleaving carbamate bonds is selected from DETA, EDA, TETA, hydrazine monohydrate, carboxylesterase, or combinations thereof.

[0174] As shown in the immobilizing agent compound of formula (IV), in some embodiments, the "linker" may include a disulfide bond. Adding a disulfide to the diimidazole carboxylate immobilizing agent compound allows for the formation of a cross-linked biomolecule of formula (IVa). As shown in scheme 6, the presence of a cross-linked biomolecule of formula (IVa) in the biological sample allows for an alternative two-step de-immobilization reaction.

[0175] Option 6

[0176]

[0177] In the first step of the reaction in Scheme 6, the cross-linked biomolecule is treated with dithiothreitol (“DTT”), a well-known reagent, which leads to the reductive cleavage of the disulfide bonds in the cross-link. DTT treatment is commonly used and readily performed under mild biological conditions. By cleaving the cross-link, DTT treatment immobilizes the biomolecule to the extent that the cross-linking stabilizes its degradation. However, the resulting de-immobilized biomolecule still retains the amine-containing moiety modified with the thiol-ethylene-carboxylic acid moiety. The urethane-modified amine moiety may interfere with certain subsequent assays of the biomolecule. For example, urethane-modified nucleic acid bases will interfere with assays utilizing reverse transcription and amplification of nucleic acid sequences. Therefore, in at least one embodiment, this disclosure provides a method in which a biological sample treated with a composition containing a formula (IV) immobilization reagent compound is contacted with a disulfide cleavage compound (e.g., DTT) and then with a urethane cleavage compound (e.g., DETA). As shown in Scheme 6, after these two treatments, the diurethane-cross-linked amine-containing moiety of the biomolecule is restored to its native state.

[0178] F. Fixed biological samples in partitions with deconsolidating agents

[0179] This document recognizes the need for methods, compositions, kits, and systems for analyzing multiple cellular analytes (e.g., genomic, epigenomic, transcriptomic, metabolomic, and / or proteomic information) from immobilized biological samples (e.g., single cells, cell populations, tissue samples, and other types of biological samples). The compositions and methods disclosed herein facilitate the preparation of biological samples reversibly immobilized with a immobilization reagent compound of formula (I), followed by provision (or encapsulation) in discrete partitions, and a decoagulating agent capable of reversing the immobilized state of the biomolecules while isolated within the partitions. Therefore, in some embodiments, this disclosure provides a method for preparing a biological sample comprising: generating discrete partitions comprising (or encapsulating) a biological sample immobilized with a diimidazole carboxylate compound of formula (I), and a decoagulating agent capable of reversing the cross-linking of molecules of formula (Ia). The method may also include an initial step of immobilizing the biological sample prior to generating the discrete partitions.

[0180] This disclosure provides methods, compositions, kits, and systems for processing immobilized biological samples to process cellular analytes. Cellular analytes suitable for use with the compositions and methods of this disclosure include, but are not limited to, intracellular and partially intracellular analytes. Cellular analytes can be proteins, metabolites, metabolic byproducts, antibodies or antibody fragments, enzymes, antigens, carbohydrates, lipids, macromolecules or combinations thereof (e.g., proteoglycans), or other biomolecules. Cellular analytes can be nucleic acid molecules. Cellular analytes can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. DNA molecules can be genomic DNA molecules. Cellular analytes can contain coding or non-coding RNA. RNA can be, for example, messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). RNA can be a transcript. RNA can be a small RNA less than 200 nucleic acid bases in length or a large RNA greater than 200 nucleic acid bases in length. Small RNAs can include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and rDNA-derived small RNA (srRNA). RNA can be double-stranded or single-stranded. RNA can be circular.

[0181] In some cases, cellular analytes are associated with intermediate entities, wherein the intermediate entities are analyzed to provide information about the cellular analytes and / or the intermediate entities themselves. For example, an intermediate entity (e.g., an antibody) may bind to a portion of an intracellular analyte (e.g., a cell surface receptor), wherein the intermediate entity is processed to provide information about the intermediate entity, the portion of the intracellular analyte, or both. In one embodiment, the intermediate entity includes an identifier (e.g., a barcode molecule) that can be used to generate barcode molecules (e.g., droplet-based barcodes), as described herein.

[0182] As used herein, the term "partition" generally refers to a space or volume suitable for accommodating one or more substances or for carrying out one or more reactions. A partition can be a physical compartment, such as a droplet or a pore (e.g., a micropore). A partition can isolate a space or volume from another space or volume. A droplet can be a first phase (e.g., an aqueous phase) in a second phase (e.g., oil) that is immiscible with the first phase. A droplet can also be a first phase in a second phase that is not separated from the first phase, such as a capsule or liposome in an aqueous phase. A partition can contain one or more other (internal) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified by an index (e.g., an index library) spanning multiple and / or remote physical compartments. For example, a physical compartment can include multiple virtual compartments.

[0183] Methods, techniques, and protocols for dividing biological samples (e.g., single cells, cellular biomolecular contents, etc.) into discrete partitions (e.g., pores or droplets) are described in this art. In one embodiment, the generated discrete partitions serve as upgrade containers, maintaining the separation of partition contents from other partition contents (e.g., droplets in an emulsion).

[0184] Methods and systems for generating stable discrete droplets containing or encapsulating individual particles from biological samples in non-aqueous or oil emulsions are described, for example, in U.S. Patent Application Publications 2010 / 0105112 and 2019 / 0100632, each of which is incorporated herein by reference for all purposes. In summary, discrete droplets in an emulsion containing or encapsulating a biological sample are achieved by introducing a flow of an aqueous fluid containing the biological sample into a flow of a non-aqueous fluid to which it is immiscible, thereby generating droplets at the junction of the two flows (see...). Figure 1-3 By providing a certain concentration and / or flow rate of water containing biological samples, the occupancy of the resulting droplets can be controlled. For example, the relative flow rates of immiscible fluids can be selected such that, on average, each discrete droplet contains less than one biological particle. Such flow rates ensure that the occupied droplets are primarily occupied by a single sample (e.g., a single cell). Discrete droplets in emulsions containing or encapsulating biological samples are also implemented using microfluidic architectures that include channel segments with channel junctions with the reservoir (see...). Figure 4-6 ).

[0185] As used herein, the term "bioparticle" generally refers to a discrete biological system derived from a biological sample. Bioparticles can be macromolecules. Bioparticles can be small molecules. Bioparticles can be viruses. Bioparticles can be cells or cell derivatives. Bioparticles can be organelles. Bioparticles can be rare cells derived from a cell population. Bioparticles can be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plant, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type, whether derived from a single-celled or multicellular organism. Bioparticles can be components of a cell. Bioparticles can be or can include DNA, RNA, organelles, proteins, or any combination thereof. Bioparticles can be obtained from the subject's tissue. Bioparticles can be sclerotic cells. Such sclerotic cells may or may not include a cell wall or cell membrane. Bioparticles may include one or more components of a cell, but may not include other components of a cell. Examples of such components are the cell nucleus or organelles.

[0186] In some cases, the droplets in multiple discrete droplets formed in this manner contain at most one type of particle (e.g., a scaffold, such as a bead) and one type of biological particle (e.g., a cell, including a fixed cell or a disintegrated cell). Flow and microfluidic channel structures can also be controlled to ensure a given number of single-occupied droplets, less than a certain level of unoccupied droplets, and / or less than a certain level of multi-occupied droplets.

[0187] In another aspect of this disclosure, fixed cells (e.g., cells fixed according to the fixation reagent composition described herein) may be partitioned with other reagents (e.g., in droplets or wells) to process one or more analytes described herein. In one embodiment, the fixed cells may be co-partitioned with a defixing agent. In another embodiment, the partition containing the fixed cells further includes a scaffold (e.g., beads) containing nucleic acid molecules suitable for barcoding one or more analytes. In another embodiment, the nucleic acid molecules may include nucleic acid sequences that provide identification information, such as barcode sequences.

[0188] As used herein, the term "barcode" generally refers to a label or identifier that conveys or is able to convey information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. In addition to endogenous characteristics of the analyte (e.g., the size of the analyte or its terminal sequences), a barcode can be a tag or combination of tags attached to an analyte (e.g., a nucleic acid molecule). A barcode can be unique. Barcodes can have many different formats. For example, a barcode can include a polynucleotide barcode; a random nucleic acid and / or amino acid sequence; and a synthetic nucleic acid and / or amino acid sequence. A barcode can be attached to an analyte in a reversible or irreversible manner. Barcodes can be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before, during, and / or after sample sequencing. Barcodes can allow the identification and / or quantification of individual sequencing reads.

[0189] As used herein, the term "barcode-encoded nucleic acid molecule" generally refers to a nucleic acid molecule produced by processing a nucleic acid barcode molecule, for example, with a nucleic acid sequence (e.g., a nucleic acid sequence complementary to the nucleic acid primer sequence covered by the nucleic acid barcode molecule). The nucleic acid sequence can be a targeted sequence (e.g., targeted by the primer sequence) or a non-targeted sequence. For example, in the methods, compositions, kits, and systems described herein, a cell's nucleic acid molecule (e.g., a messenger RNA (mRNA) molecule) is hybridized with a nucleic acid barcode molecule (e.g., a nucleic acid barcode molecule containing a barcode sequence and a nucleic acid primer sequence complementary to the nucleic acid sequence of the mRNA molecule) and reverse transcribed to produce a barcode nucleic acid molecule having a sequence corresponding to the mRNA nucleic acid sequence and a barcode sequence (or its reverse complementary sequence). The barcode nucleic acid molecule can be used as a template, such as a template polynucleotide, which can be further processed (e.g., amplified) and sequenced to obtain a targeted nucleic acid sequence. For example, in the methods and systems described herein, a barcode-encoded nucleic acid molecule can be further processed (e.g., amplified) and sequenced to obtain a nucleic acid sequence of the mRNA.

[0190] As used herein, the term "bead" generally refers to a particle. Beads can be solid or semi-solid particles. Beads can be gel beads. Gel beads can include a polymer matrix (e.g., a matrix formed by polymerization or crosslinking). The polymer matrix can include one or more polymers (e.g., polymers with different functional groups or repeating units). The polymers in the polymer matrix can be randomly arranged, as in random copolymers, and / or have an ordered structure, as in block copolymers. Crosslinking can occur via covalent, ionic or induced, interaction or physical entanglement. Beads can be macromolecules. Beads can be formed from nucleic acid molecules bonded together. Beads can be formed via covalent or non-covalent assembly of molecules such as monomers or polymers (e.g., macromolecules). Such polymers or monomers can be natural or synthetic. Such polymers or monomers can be or include, for example, nucleic acid molecules (e.g., DNA or RNA). Beads can be formed from polymer materials. Beads can be magnetic or non-magnetic. Beads can be rigid. Beads can be flexible and / or compressible. Beads can be destructible or soluble. Beads can be solid particles (e.g., metal-based particles, including but not limited to iron oxide, gold, or silver) covered with a coating containing one or more polymers. Such coatings can be destructible or soluble.

[0191] Figure 1An exemplary microfluidic channel structure 100 for generating discrete droplets containing or encapsulating particles from a biological sample (e.g., a single cell) is shown. Channel structure 100 may include channel segments 102, 104, 106, and 108 communicating at a channel junction 110. In operation, a first aqueous fluid 112 comprising suspended particles (e.g., cells) from a biological sample 114 is delivered along channel segment 102 to junction 110, while a second fluid 116 (or “partitioned fluid”) immiscible with the aqueous fluid 112 is delivered from each of channel segments 104 and 106 to junction 110 to form discrete droplets 118, with 120 of the first aqueous fluid 112 flowing into channel segment 108 and out of junction 110. Channel segment 108 may be fluidly coupled to an outlet reservoir in which these discrete droplets may be stored and / or harvested. The generated discrete droplets may include individual particles (such as droplet 118) from biological sample 114, or may include discrete droplets comprising more than one particle 114. Figure 1 (Not shown in the image). Discrete droplets may not contain biological particles 114 (such as droplet 120). Each discrete droplet is able to maintain the separation of its own contents (e.g., individual biological sample particles 114) from the contents of other droplets.

[0192] Typically, the second fluid 116 comprises an oil, such as a fluorinated oil, which includes a fluorinated surfactant that helps stabilize the resulting droplets. Examples of useful partitioned fluids and fluorinated surfactants are described, for example, in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference for all purposes.

[0193] like Figure 1 The microfluidic channel shown for generating discrete droplets can be coupled to any of a variety of fluid source or receiving components, including reservoirs, pipes, manifolds, or other fluid components of a system. Furthermore, the microfluidic channel structure 100 can have other geometries, including those with more than one channel junction. For example, the microfluidic channel structure can have 2, 3, 4, or 5 channel segments, each carrying biological sample particles, assay reagents, and / or beads that meet at the channel junction.

[0194] Typically, the fluid used to generate discrete droplets is directed to flow through one or more fluid flow units along one or more channels or reservoirs. The fluid flow units may include compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, etc., to control the flow of the fluid. The fluid can also be controlled, or otherwise, by applied pressure differentials, centrifugal force, electric pumps, vacuum, capillary action, or gravity flow.

[0195] Those skilled in the art will recognize that many different microfluidic channel designs can be used in conjunction with the methods and compositions of this disclosure to provide discrete droplets containing biological sample particles immobilized with a compound of formula (I), which is a decoupling agent capable of cleaving bis(urethane) crosslinks (e.g., DETA), and / or beads with barcodes and / or other assay reagents.

[0196] Including barcodes in discrete partitions (e.g., wells or droplets) and providing a unique identifier for the biological sample allows for the differentiation and individual analysis of data from the biological sample. Barcodes can be delivered in the discrete partitions before, after, or simultaneously with the biological sample. For example, barcodes can be injected into droplets before, after, or simultaneously with droplet generation. Barcodes that can be used in the methods and compositions of this disclosure typically contain nucleic acid molecules (e.g., oligonucleotides). Nucleic acid barcode molecules are typically delivered to the partitions via a scaffold (such as beads). In some cases, the barcode nucleic acid molecules are initially associated with the beads when the discrete partitions are provided (e.g., discrete wells are provided or discrete droplets are generated), and then released from the beads upon application of a stimulus to the partition (e.g., a well or droplet). Barcode-carrying beads that can be used in the methods and compositions of this disclosure are described in further detail elsewhere herein.

[0197] Methods and systems for partitioning barcode-carrying beads into droplets are provided in U.S. Patent Nos. 1,048,0029, 1,085,8702, and 1,072,5027, U.S. Patent Publications Nos. 2019 / 0367,997 and 2019 / 0064,173, and International Applications Nos. PCT / US20 / 17785 and PCT / US20 / 020486, each of which is incorporated herein by reference in its entirety for all purposes.

[0198] Figure 8 An embodiment of a bead carrying a barcode is illustrated. A nucleic acid molecule 802 (e.g., an oligonucleotide) can be coupled to a bead 804 via a releasable linker 806 (e.g., a disulfide linker). The same bead 804 can be coupled (e.g., via a releasable linker) to one or more other nucleic acid molecules 818, 820. The nucleic acid molecule 802 may be or contain a barcode. As described elsewhere herein, the structure of the barcode may include a number of sequence elements. The nucleic acid molecule 802 may contain a functional sequence 808 that can be used for subsequent processing. For example, the functional sequence 808 may include one or more sequencer-specific flow cell attachment sequences (e.g., for...). The P5 sequence of the sequencing system and the sequencing primer sequences (e.g., for sequencing systems) The sequencing system's R1 primer. Nucleic acid molecule 802 may contain a barcode sequence 810 for barcoding a sample (e.g., DNA, RNA, protein, antibody, etc.). In some cases, the barcode sequence 810 may be bead-specific, such that the barcode sequence 810 is common to all nucleic acid molecules (e.g., including nucleic acid molecule 802) coupled to the same bead 804. Alternatively or additionally, the barcode sequence 810 may be partition-specific, such that the barcode sequence 810 is common to all nucleic acid molecules coupled to one or more beads separated into the same partition. Nucleic acid molecule 802 may contain a specific initiation sequence 812, such as an mRNA-specific initiation sequence (e.g., a poly-T sequence), a targeted initiation sequence, and / or a random initiation sequence. Nucleic acid molecule 802 may contain an anchoring sequence 814 to ensure that the specific initiation sequence 812 hybridizes at the sequence ends (e.g., of mRNA). For example, anchor sequence 814 may include random short nucleotide sequences, such as 1-mer, 2-mer, 3-mer, or longer sequences, which can ensure that poly-T fragments are more likely to hybridize at the end of the poly-A tail of the mRNA.

[0199] Nucleic acid molecule 802 may contain a unique molecular recognition sequence 816 (e.g., a unique molecular identifier (UMI)). In some cases, the unique molecular recognition sequence 816 may contain about 5 to about 8 nucleotides. Alternatively, the unique molecular recognition sequence 816 may be compressed to less than about 5 or more than about 8 nucleotides. The unique molecular recognition sequence 816 may be a unique sequence that varies on individual nucleic acid molecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g., bead 804). In some cases, the unique molecular recognition sequence 816 may be a random sequence (e.g., a random N-mer sequence). For example, a UMI can provide a unique identifier for the captured initiating mRNA molecule to allow for quantification of the amount of originally expressed RNA. As will be understood, although Figure 8 Three nucleic acid molecules, 802, 818, and 820, are shown coupled to the surface of bead 804; however, a single bead can be coupled to any number of individual nucleic acid molecules, for example, from one to hundreds of thousands or even millions of individual nucleic acid molecules. The corresponding barcode for each individual nucleic acid molecule may contain common or relatively common sequence fragments (e.g., 808, 810, 812, etc.) and variable or unique sequence fragments (e.g., 816) between different individual nucleic acid molecules coupled to the same bead.

[0200] Biological particles (e.g., cells, fixed cells, deconsolidated cells, DNA, RNA, etc.) can co-partition with barcode-carrying beads 804. Barcode-encoded nucleic acid molecules 802, 818, and 820 can be released from beads 804 within the partition. For example, in the context of analyzing sample RNA, a poly-T fragment (e.g., 812) of one of the released nucleic acid molecules (e.g., 802) can hybridize with the poly-A tail of the mRNA molecule. Reverse transcription produces a cDNA transcript of the mRNA, but this transcript includes each of the sequence fragments 808, 810, and 816 of nucleic acid molecule 802. Because nucleic acid molecule 802 contains the anchoring sequence 814, it is more likely to hybridize with the sequence terminus of the poly-A tail of the mRNA and initiate reverse transcription. Within any given partition, all cDNA transcripts of individual mRNA molecules may include a common barcode sequence fragment 810.

[0201] However, transcripts made from different mRNA molecules within a given partition can vary at unique molecular recognition sequence fragments (e.g., UMI fragments). Advantageously, even after any subsequent amplification of the contents of a given partition, the number of different UMIs can indicate the amount of mRNA originating from the given partition and thus from biological particles (e.g., cells, fixed cells, deconsolidated cells, etc.). As described above, transcripts can be amplified, cleaned, and sequenced to identify the cDNA transcript sequence of the mRNA, as well as the barcode fragments and UMI fragments can be sequenced. Although poly-T primer sequences are described, other targeted or random priming sequences can also be used to initiate reverse transcription reactions. Similarly, although described as releasing barcode-encoded oligonucleotides into the partition, in some cases, nucleic acid molecules bound to beads (e.g., gel beads) can be used to hybridize and capture mRNA on the solid phase of the beads, for example, to facilitate the separation of RNA from other cellular contents. In such cases, further processing can be performed either within or outside the partition (e.g., in batches). For example, RNA molecules on the beads can be reverse transcribed or subjected to other nucleic acid treatments, additional ligant sequences can be added to barcode-encoded nucleic acid molecules, or other nucleic acid reactions (e.g., amplification, nucleic acid extension) can be performed. The beads or their products (e.g., barcode-encoded nucleic acid molecules) can be collected from partitions and / or pooled together and subsequently cleaned and further characterized (e.g., sequencing). The operations described herein can be performed at any useful or convenient step. For example, beads containing nucleic acid barcode molecules can be introduced into partitions (e.g., wells or droplets) before, during, or after the sample is introduced into the partitions. Nucleic acid molecules from the sample can be barcode-encoded, which can occur on the beads (where nucleic acid molecules remain coupled to the beads) or after the nucleic acid barcode molecules are released into the partitions. Where nucleic acid molecules from the sample remain attached to the beads, beads from various partitions can be collected, pooled, and further processed (e.g., reverse transcription, ligant attachment, amplification, cleanup, sequencing). In other cases, processing can occur within the partitions. For example, conditions can be provided in the partitions sufficient for barcode encoding, ligand attachment, reverse transcription, or other nucleic acid processing operations, and these operations can be performed before cleaning and sequencing.

[0202] Figure 9Another embodiment of a bead carrying a barcode is illustrated. A nucleic acid molecule 905, such as an oligonucleotide, can be coupled to a bead 904 via a releasable bond 906 (e.g., a disulfide linker). The nucleic acid molecule 905 may contain a first capture sequence 960. The same bead 904 can be coupled (e.g., via a releasable bond) to one or more other nucleic acid molecules 903, 907 containing other capture sequences. The nucleic acid molecule 905 may be or contain a barcode. As described elsewhere herein, the barcode structure may contain a number of sequence elements, such as functional sequences 908 (e.g., flow cell attachment sequences, sequencing primer sequences, etc.), barcode sequences 910 (e.g., bead-specific sequences common to the bead, region-specific sequences common to different regions, etc.), and unique molecular identifiers 912 (e.g., unique sequences attached to different molecules within the bead) or portions thereof. The capture sequence 960 can be configured to attach to a corresponding capture sequence 965. In some cases, the corresponding capture sequence 965 can be coupled to another molecule, which can be an analyte or an intermediate carrier. For example, as Figure 9 As illustrated, the corresponding capture sequence 965 is coupled to a guide RNA molecule 962 containing a target sequence 964, wherein the target sequence 964 is configured to attach to the analyte. Another oligonucleotide molecule 907 attached to bead 904 contains a second capture sequence 980, which is configured to attach to a second corresponding capture sequence 985. Figure 9 As illustrated, the second corresponding capture sequence 985 is coupled to antibody 982. In some cases, antibody 982 may have binding specificity for the analyte (e.g., surface protein). Alternatively, antibody 982 may not have binding specificity. Another oligonucleotide molecule 903 attached to bead 904 contains a third capture sequence 970, which is configured to attach to the second corresponding capture sequence 975. Figure 9 As illustrated, the third corresponding capture sequence 975 is coupled to molecule 972. Molecule 972 may or may not be configured as a targeted analyte. Other oligonucleotide molecules 903, 907 may contain other sequences described with respect to oligonucleotide molecule 905 (e.g., functional sequences, barcode sequences, UMIs, etc.). Although in Figure 9The diagram illustrates a single oligonucleotide molecule containing each capture sequence; however, it should be understood that for each capture sequence, the bead may contain a set of one or more oligonucleotide molecules, each containing the capture sequence. For example, the bead may contain any number of sets or sets of different capture sequences. Alternatively or additionally, bead 904 may contain other capture sequences. Alternatively or additionally, bead 904 may contain fewer types of capture sequences (e.g., two capture sequences). Optionally or additionally, bead 904 may contain oligonucleotide molecules containing promoter sequences, such as specific promoter sequences, such as mRNA-specific promoter sequences (e.g., poly-T sequences), targeted promoter sequences, and / or random promoter sequences, to facilitate the assay of gene expression.

[0203] Figure 2An exemplary microfluidic channel structure 200 for generating discrete droplets is shown, comprising or encapsulating beads 214 carrying barcodes and biosample particles 216. Channel structure 200 includes channel segments 201, 202, 204, 206, and 208 fluidly connected at a channel junction 210. In operation, channel segment 201 transports an aqueous fluid 212, which may include a plurality of beads 214 (e.g., gel beads carrying barcode oligonucleotides), along channel segment 201 to junction 210. The plurality of beads 214 may be derived from a suspension of beads. For example, channel segment 201 may be connected to a reservoir containing an aqueous suspension of beads 214. Channel segment 202 transports an aqueous fluid 212, which includes a plurality of biosample particles 216, along channel segment 202 to junction 210. The plurality of biosample particles 216 may be derived from a suspension of biosample particles. For example, channel segment 202 may be connected to a reservoir containing an aqueous suspension of biosample particles 216. In some cases, the aqueous fluid 212 in one or both segments of the first channel segment 201 or the second channel segment 202 may include one or more reagents, as described elsewhere herein. For example, in some embodiments of this disclosure, where the biosample particles are immobilized with a compound of formula (I), the aqueous fluid in the first and / or second channel segments, respectively delivering the biosample and the beads, may include a deconsolidating agent capable of cleaving diurethane crosslinks (e.g., DETA). A second fluid 218, immiscible with the aqueous fluid 212, is delivered from each of channel segments 204 and 206 to junction 210. When an aqueous fluid 212 from one of channel sections 201 and 202 and a second fluid 218 (e.g., fluorinated oil) from one of channel sections 204 and 206 meet at channel junction 210, the aqueous fluid 212 is partitioned into discrete droplets 220 in the second fluid 218 and flows away from junction 210 along channel section 208. Channel section 208 can then deliver discrete droplets containing or encapsulating biological sample particles and barcode-carrying beads to an outlet reservoir fluidly coupled to channel section 208, where these droplets can be collected.

[0204] Alternatively, channel segments 201 and 202 may meet at another junction upstream of junction 210. At such junction, beads and bioparticles can form a mixture that is guided along another channel to junction 210 to produce droplets 220. The mixture can be provided with beads and bioparticles in an alternating manner, such that, for example, the droplet contains a single bead and a single bioparticle.

[0205] Use such as Figure 2The channel system illustrated herein can generate discrete droplets 220 containing or encapsulating individual bioparticles and a bead of a biological sample, wherein the bead may carry a barcode and / or another reagent. It has also been considered that, in some cases, the use of... Figure 2 The channel system generates discrete droplets, wherein the droplets may include more than one individual biological sample particle or may not include a biological sample. Similarly, in some embodiments, the discrete droplets may include more than one bead or may not include a bead. The discrete droplets may also be completely unoccupied (e.g., without a bead or biological sample).

[0206] In some embodiments, it is desirable for the beads, biological sample particles, and generated discrete droplets to flow along a channel at a substantially regular flow rate, generating discrete droplets containing individual beads and individual biological sample particles. Conventional flow rates and devices that can be used to provide such conventional flow rates are known in the art, see, for example, U.S. Patent Publication No. 2015 / 0292988, which is incorporated herein by reference in its entirety. In some embodiments, the flow rate is set to provide discrete droplets containing individual beads and biological sample particles with yields greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0207] G. The purpose of the brackets provided in the partition

[0208] The scaffold may carry barcodes and / or other reagents useful for the compositions and methods of this disclosure, and may include, but is not limited to, porous, non-porous, solid, semi-solid, semi-fluid, fluid beads and / or combinations thereof. In some embodiments, the beads may be made of soluble, destructible, and / or degradable materials, such as gel beads comprising hydrogels. Alternatively, in some embodiments, the beads are non-degradable.

[0209] In some embodiments of this disclosure, beads are provided in discrete partitions (e.g., beads are provided or encapsulated in discrete droplets or provided in discrete pores), with the biological sample being the beads. Typically, beads used in the embodiments disclosed herein comprise hydrogels. Such gel beads can be formed from molecular precursors, such as polymers or monomeric substances, which undergo reactions to form cross-linked gel polymers. Another type of semi-solid bead that can be used in this disclosure is liposome beads. In some embodiments, the beads used can be solid beads comprising metals, including iron oxide, gold, and silver. In some cases, the beads can be silica beads. In some cases, the beads can be rigid. In other cases, the beads can be flexible and / or compressible. Typically, the beads can be any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, rectangular, amorphous, circular, cylindrical, and variations thereof.

[0210] The multiple beads used in the embodiments may have a uniform size, or they may comprise a collection of different sizes. In some cases, the diameter of the beads is at least about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1000 μm (1 mm) or larger. In some cases, the diameter of the beads may be less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or smaller. In some cases, the diameter of the beads may be in the range of approximately 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

[0211] In some embodiments, the beads used are a group or multiple beads having a relatively monodisperse size distribution. Generally, if it is desired to provide a consistent amount of reagent within discrete partitions (e.g., pores or droplets), using relatively consistent bead characteristics (e.g., size) can provide overall consistency of the contents of each partition. For example, beads that can be used in embodiments of this disclosure may have a size distribution with a coefficient of variation of their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or even smaller.

[0212] Beads that can be used in the methods and compositions of this disclosure may comprise a range of natural and / or synthetic materials. For example, beads may comprise natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid (DNA), rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, psyllium husk, gum arabic, agar, gelatin, shellac, syzygium aromaticum, xanthan gum, corn gum, guar gum, arabic gum, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylic acid, nylon, silicone, spandex, viscose rayon, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(trifluorochloroethylene), poly(ethylene oxide), polyethylene terephthalate, polyethylene, polyisobutylene, poly(methyl methacrylate), poly(formaldehyde), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), and / or combinations thereof (e.g., copolymers). Beads can also be formed from materials other than polymers, including lipids, micelles, ceramics, glass ceramics, material composites, metals, and other inorganic materials.

[0213] although Figure 1 and Figure 2 While the provision of substantially single-occupied discrete droplets has been described, in some embodiments it is also contemplated that it is desirable to provide multiple-occupied discrete droplets, such as single droplets comprising two, three, four, or more cells from a biological sample, and / or multiple distinct beads, such as beads carrying barcoded nucleic acid molecules and / or beads carrying reagents (e.g., lysis agents, disintegrating agents, and / or assay reagents). Therefore, as described elsewhere herein, the flow characteristics of the biological particles and / or beads can be controlled to provide such multi-occupied droplets. In particular, the flow parameters of the liquid used in the channel structure can be controlled to provide a given droplet occupancy rate greater than about 50%, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.

[0214] In some embodiments, the beads useful in the compositions and methods of this disclosure are those capable of delivering reagents (e.g., dissolving agents and / or assay reagents) into discrete partitions (e.g., droplets) containing biological sample particles that have been treated with a compound of formula (I) to fix them. In some embodiments, different beads (e.g., containing different reagents) can be introduced from different sources into different inlets leading to a common droplet-generating junction (e.g., junction 210). In such cases, the flow rate and frequency of the different beads entering the channel or junction can be controlled to provide a specific ratio of beads from each source while ensuring that a given pair or combination of such beads forms a partition with a given number of biological particles (e.g., one biological particle and one bead per partition).

[0215] The discrete droplets described herein typically comprise small volumes, such as less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less. In some embodiments, the total volume of the generated discrete droplets containing or encapsulating biological particles from a sample is less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, or less. It should be understood that the volume of sample fluid within the droplet, including co-located biological particles and / or beads, may be less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the aforementioned volume.

[0216] Methods for generating discrete droplets useful to the compositions and methods of this disclosure result in the generation of a population or plurality of discrete droplets comprising biological sample particles (e.g., biological samples immobilized with a compound of formula (I)) and other reagents (e.g., destabilizing agents, such as DETA). Typically, these methods are readily controllable to provide any suitable number of droplets. For example, at least about 1,000 discrete droplets, at least about 5,000 discrete droplets, at least about 10,000 discrete droplets, at least about 50,000 discrete droplets, at least about 100,000 discrete droplets, at least about 500,000 discrete droplets, at least about 1,000,000 discrete droplets, at least about 5,000,000 discrete droplets, at least about 10,000,000 discrete droplets, or more discrete droplets can be generated. Furthermore, the plurality of discrete droplets may comprise unoccupied droplets and occupied droplets.

[0217] As described elsewhere herein, in some embodiments of the compositions and methods disclosed herein, the generated discrete droplets comprising or encapsulating biosample particles and optionally one or more different types of beads further contain other reagents. In some embodiments, the other reagents contained in or encapsulated in the droplets include lysing agents and / or destabilizing agents, which function to release and / or decapsulate the biomolecular contents of the biosample particles within the droplets. In some embodiments, the lysing agent and / or destabilizing agent may be contacted with the biosample suspension simultaneously with or prior to introducing the biosample particles into the droplet-generating junction (e.g., junction 210) of the microfluidic system. In some embodiments, the reagents are introduced via one or more additional channels upstream of the channel junction.

[0218] In some implementations, biological sample particles can be co-located with other reagents. Figure 3An embodiment of a microfluidic channel structure 300 for co-dividing biological sample particles and other reagents (including lysing agents and / or destabilizing agents) is shown. The channel structure 300 may include channel segments 301, 302, 304, 306, and 308. Channel segments 301 and 302 are connected at a first channel junction 309. Channel segments 302, 304, 306, and 308 are connected at a second channel junction 310. In an exemplary co-dividing operation, channel segment 301 may transport an aqueous fluid 312 comprising a plurality of biological sample particles 314 (e.g., immobilized biological samples) along channel segment 301 to the second junction 310. Alternatively or supplementarily, channel segment 301 may transport beads (e.g., gel beads carrying barcodes). For example, channel segment 301 may be connected to a reservoir containing an aqueous suspension of biological sample particles 314. Upstream of and immediately before reaching the second junction 310, channel segment 301 may meet channel segment 302 at the first junction 309. Channel segment 302 may transport multiple reagents 315 (e.g., lysis agents or dissolving agents) in the aqueous fluid 312 to the first junction 309. For example, channel segment 302 may be connected to a reservoir including reagents 315. After the first junction 309, the aqueous fluid 312 in channel segment 301 may carry biological sample particles 314 and reagents 315 to the second junction 310. In some cases, the aqueous fluid 312 in channel segment 301 may include one or more reagents, which may be the same as or different from reagents 315. A second fluid 316 (e.g., fluorinated oil) immiscible with the aqueous fluid 312 may be delivered from each of channel segments 304 and 306 to the second junction 310. When the aqueous fluid 312 from channel segment 301 and the second fluid 316 from each of channel segments 304 and 306 meet at the second channel junction 310, the aqueous fluid 312 is partitioned into discrete droplets 318 in the second fluid 316 and flows away from the second junction 310 along channel segment 308. Channel segment 308 can deliver the discrete droplets 318 to an outlet reservoir fluidly coupled to channel segment 308, where they can be collected for further analysis.

[0219] The generated discrete droplets may include individual biological sample particles 314 and / or one or more reagents 315, depending on what reagents are included in the channel segment 302. In some cases, the generated discrete droplets may also include beads carrying barcodes (not shown), as can be added via other channel structures described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., no reagents, no biological particles). Typically, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including fluid components of reservoirs, conduits, manifolds, or other systems. It should be understood that the microfluidic channel structure 300 may have other geometries. For example, the microfluidic channel structure may have more than two channel junctions. For example, the microfluidic channel structure may have 2, 3, 4, 5, or more channel segments, each carrying the same or different types of beads, reagents, and / or biological sample particles that meet at the channel junctions. Fluid flow in each channel segment can be controlled to control the partitioning of different elements into droplets. Fluids can be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit may include a compressor (e.g., providing positive pressure), a pump (e.g., providing negative pressure), an actuator, etc., to control the flow of fluids. Fluids can also be controlled, or otherwise, by applied pressure differentials, centrifugal force, electric pumps, vacuum, capillary action, or gravity flow.

[0220] Figure 4 An embodiment of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets is illustrated. The channel structure 400 may include a channel segment 402 communicating with a reservoir 404 at a channel junction 406 (or intersection). The reservoir 404 may be a chamber. As used herein, any reference to “reservoir” may also refer to a “chamber.” In operation, an aqueous fluid 408 containing suspended beads 412 may be conveyed along the channel segment 402 to the junction 406 to meet with a second fluid 410 immiscible with the aqueous fluid 408 in the reservoir 404 to generate droplets 416, 418 of the aqueous fluid 408 flowing into the reservoir 404. At the junction 406, the aqueous fluid 408 and the second fluid 410 meet, and droplets may form based on factors such as the hydrodynamics at the junction 406, the flow rates of the two fluids 408, 410, fluid properties, and certain geometric parameters of the channel structure 400 (e.g., w, h0, α, etc.). Multiple droplets can be collected in reservoir 404 by continuously injecting aqueous fluid 408 from channel section 402 through junction 406.

[0221] Figure 5An embodiment of a microfluidic channel structure for increasing droplet generation flux is illustrated. The microfluidic channel structure 500 may include a plurality of channel segments 502 and a reservoir 504. Each of the plurality of channel segments 502 may be in fluid communication with the reservoir 504. The channel structure 500 may include a plurality of channel junctions 506 between the plurality of channel segments 502 and the reservoir 504. Each channel junction can be a droplet generation point. Figure 4 Any description of channel segment 402 and its components in channel structure 400 may correspond to any description of a given channel segment 502 and its corresponding components in channel structure 500. Any description of storage 404 and its components from channel structure 400 may correspond to any description of storage 504 and its corresponding components from channel structure 500.

[0222] Figure 6 Another embodiment of a microfluidic channel structure for increasing droplet generation flux is shown. The microfluidic channel structure 600 may include a plurality of channel segments 602 arranged generally in a circular pattern around the periphery of a reservoir 604. Each of the plurality of channel segments 602 may be in fluid communication with the reservoir 604. The channel structure 600 may include a plurality of channel junctions 606 between the plurality of channel segments 602 and the reservoir 604. Each channel junction can be a droplet generation point. Figure 4 Any description of channel segment 402 and its components in channel structure 400 may correspond to any description of a given channel segment 602 and its corresponding components in channel structure 600. Any description of reservoir 404 and its components from channel structure 400 may correspond to any description of reservoir 604 and its corresponding components from channel structure 600. Additional aspects of such microfluidic structures, including systems and methods for implementing them, are provided in U.S. Patent Application Publication No. 20190323088, the entire contents of which are incorporated herein by reference.

[0223] Once the lysing agent and / or destabilizing agent co-partitions with the biosample particles immobilized by the compound of formula (I) in partitions (e.g., pores or droplets), these agents facilitate the release and destabilization of the biomolecular contents of the biosample particles within the partitions. As described elsewhere herein, the destabilized biomolecular contents released in one partition remain discrete from the contents of other partitions, thereby allowing for the detection and quantification of target biomolecular analytes present in the different biosamples.

[0224] Examples of lysing agents that can be used in the compositions and methods of this disclosure include bioactive agents such as lysins for lysing different cell types (e.g., Gram-positive or Gram-negative bacteria, plants, yeast, mammals, etc.), such as lysozyme, colorless peptidase, lysostaphin, lipase, cell lysin, cytolysin, and other lysins available from, for example, Sigma-Aldrich (St. Louis, Missouri), and other commercially available lysins. Other lysins may additionally or alternatively co-partition with biological particles to release the contents of the biological sample into partitions (e.g., pores or droplets). For example, in some cases, surfactant-based lysins may be used to lyse cells, although these may be less desirable for emulsion-based systems where surfactants can interfere with emulsion stabilization. In some embodiments, the lysin may include nonionic surfactants such as, for example, Triton X-100 and Tween 20. In some cases, the lysin may include ionic surfactants such as sodium dodecyl sarcosinate and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic, or mechanical cell disruption can also be used in certain situations, such as the provision or encapsulation of biological particles that can supplement or replace droplet partitions, based on non-emulsion-based partitions, where any pore size of the encapsulation is small enough to retain nucleic acid fragments of a given size after cell disruption.

[0225] In addition to lysing agents and / or deconsolidating agents that co-disperse with biological sample particles into discrete partitions (e.g., pores or droplets), it is further conceivable that other assay reagents can also co-disperse within these partitions. Examples include DNase and RNase inactivators or inhibitors, such as proteinase K; chelating agents, such as EDTA; and other reagents used to remove or otherwise reduce the negative activity or influence of different cell lysate components on subsequent nucleic acid processing.

[0226] In some embodiments, biosample particles, provided or encapsulated in discrete partitions (e.g., pores or droplets) along with other reagents, are exposed to appropriate stimuli to release the biomolecular contents of the sample particles and / or the contents of co-partitioned beads. For example, in some embodiments, a chemical stimulant may be co-partitioned within the partitions along with the biosample particles and beads (e.g., gel beads) to allow the beads to degrade and release their contents into the partitions. In some embodiments, discrete partitions (e.g., capable of generating droplets) and a disintegrating agent immobilizing the biosample particles with a compound of formula (I) and capable of cleaving diurethane crosslinks (e.g., DETA) may be provided, wherein the disintegrating agent is contained in beads (e.g., gel beads) that are degradable by thermal stimulation. In such embodiments, the partitions are exposed to thermal stimulation, thereby causing the beads to degrade and release the disintegrating agent. In another embodiment, a partition is envisioned comprising (e.g., provided or encapsulated droplets) biological sample particles immobilized with a compound of formula (I) and two distinct beads (e.g., one bead carrying a disintegrating agent and the other carrying an assay reagent), wherein the contents of the two distinct beads are released by non-overlapping stimuli (e.g., chemical and thermal stimuli). Such an embodiment allows for the release of different reagents into the same discrete partition at different times. For example, a first bead triggered by a thermal stimulus releases the disintegrating agent into the partition, and then, after a set time, a second bead triggered by a chemical stimulus releases the assay reagent, detecting the analyte in the biological sample particles disintegrated by contact with the disintegrating agent.

[0227] Other assay reagents can also be co-divided into discrete partitions (e.g., wells or droplets) with the biological sample, such as endonucleases for fragmenting DNA from the biological sample, DNA polymerases for amplifying nucleic acid fragments from the biological sample and attaching barcode molecular tags to the amplified fragments, and dNTPs. Other enzymes can be co-divided, including but not limited to polymerases, transposases, ligases, proteinase K, DNase, etc. Other assay reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides that can be used for template switching (also referred to herein as "switch oligonucleotides" or "template-switching oligonucleotides").

[0228] In some embodiments, template switching can be used to increase the length of the cDNA generated during assay. In some embodiments, template switching can be used to attach a predetermined nucleic acid sequence to the cDNA. In embodiments of template switching, cDNA can be generated from a template, such as cellular mRNA, through reverse transcription, wherein a reverse transcriptase with terminal transferase activity can add additional nucleotides, such as polyC, to the cDNA in a template-independent manner.

[0229] Once the contents of a biological sample's cells are released into discrete partitions (e.g., pores or droplets), the biomolecular components contained therein (e.g., macromolecular components of the biological sample, such as RNA, DNA, or proteins) can be further processed within the partition. According to the methods and systems described herein, unique barcode identifiers can be provided for the biomolecular contents of individual biological samples, and they can be attributed to the same biological sample when characterizing biomolecular components (e.g., in sequencing assays). The ability to attribute characteristics to individual biological samples or groups of biological samples is provided by specifically assigning nucleic acid barcode sequences to individual biological samples or groups of biological samples.

[0230] In some embodiments, a unique identifier barcode is provided in the form of a nucleic acid molecule (e.g., an oligonucleotide), which includes sequences that can be attached to or otherwise associated with nucleic acid contents or other components of a single biological sample. In some embodiments, only one nucleic acid barcode sequence is associated with a given discrete partition (e.g., a pore or droplet), although in some cases, two or more different barcode sequences may exist. The nucleic acid barcode sequence may comprise about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecule (e.g., an oligonucleotide). In some cases, the length of the barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In other cases, the length of the barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely continuous, i.e., in a single adjacent nucleotide segment, or they can be separated into two or more separate subsequences separated by one or more nucleotides. In some cases, the length of the separated barcode subsequences can be from about 4 to about 16 nucleotides. In some cases, the barcode sequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0231] In some embodiments, the nucleic acid barcode molecule may also include other functional sequences for processing nucleic acids from biological samples (e.g., wells or droplets) within the partition. These functional sequences may include, for example, targeted or random / universal amplification primer sequences for amplifying nucleic acid molecules from individual biological samples within the partition while attaching a combined barcode sequence, sequencing primers or primer recognition sites, hybridization or probe sequences, such as those for recognizing the presence of a sequence or for pulling down the barcode-encoded nucleic acid molecule, or any of many other potential functional sequences.

[0232] In some embodiments, a large number of nucleic acid barcode molecules (e.g., oligonucleotides) are releaseably attached to the beads, wherein all nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but a large number of different barcode sequences are presented in the population of beads used. In some embodiments, as described elsewhere herein, gel beads (e.g., comprising a polyacrylamide polymer matrix) serve as a solid scaffold and delivery carrier for delivering nucleic acid molecules into partitions because they are capable of carrying a large number of nucleic acid molecules and can be configured to release those nucleic acid molecules upon exposure to a specific stimulus. In some cases, the bead population provides different barcode sequence libraries containing at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences or more.

[0233] Nucleic acid barcode molecules can be released from the beads upon application of specific stimuli. In some cases, the stimulus can be light stimulation, for example, by cleaving the photoinstantaneous bonds of the releasing nucleic acid molecules. In other cases, thermal stimulation can be used, where raising the temperature of the bead environment will cause bond cleavage or other release of nucleic acid molecules from the beads. In still other cases, chemical stimuli can be used to cleave the bonds between the nucleic acid molecules and the beads or otherwise cause the release of nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrix described above for providing or encapsulating biological samples and can be degraded by exposure to a reducing agent (such as DTT) to release attached nucleic acid molecules.

[0234] H. Use of fixed biological samples and deconsolidation agents in zonal-based assays

[0235] As disclosed elsewhere herein, the compositions and methods of this disclosure allow for the preparation of biological samples immobilized with a bis-imidazolium-carboxylate compound of formula (I), which is provided in discrete partitions (e.g., in discrete wells, or in discrete droplets, or encapsulated (optionally, as a single cell)), optionally together with a lysing agent and / or a deconsolidating agent capable of reversing the crosslinking of the sample with the bis-carbamate, thereby allowing the determination of cellular analytes of the sample as if they were obtained from fresh biological samples. In one embodiment, a single cell (e.g., a single immobilized cell) is provided in a discrete partition. The immobilization reagent compounds, methods, and associated deconsolidating agents of this disclosure allow for the immediate preservation of fresh biological samples, followed by storage for a period of time before being provided with the lysing agent and / or deconsolidating agent in a partition (e.g., provided in a well, or provided in a droplet, or encapsulated in a droplet). Typically, other materials, such as unique nucleic acid barcode molecules and assay reagents (e.g., provided in a well, or provided in a droplet, or encapsulated), are also provided in the partition. Therefore, it is conceivable that the method of this disclosure can be implemented in a manner in which the amount of time between immobilizing a biological sample with a bis-imidazolium-carboxylate compound of formula (I) and providing the sample for treatment in discrete partitions (e.g., in wells, in droplets, or by encapsulation in discrete droplets) is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 72 hours, at least 1 week, at least 1 month, at least 6 months, or longer.

[0236] Generally, it is contemplated that any method for preparing a biological sample using a fixation reagent composition comprising a compound of formula (I) disclosed herein (including formulas (II), (III), (IV), (V) or compound (6)) can also be used to prepare a fixed biological sample provided in a partition (e.g., provided in a well or encapsulated in a droplet), optionally, together with a lysis agent and / or a destabilizing agent. Similarly, it is contemplated that any composition disclosed herein comprising a diurethane cross-linked biomolecule of formula (Ia) (including compounds of formulas (Iia), (IIIa), (Iva), (Va), or (Via)) can be provided in a partition with a destabilizing agent (e.g., provided in a well or encapsulated in a droplet) and used in partition-based assay methods (e.g., well-based or droplet-based assay methods). Thus, in at least one embodiment, this disclosure provides an assay method comprising: (a) generating a discrete partition comprising a fixed biological sample (a well or droplet containing or encapsulating the fixed biological sample), a lysis agent and / or a destabilizing agent, and an analytical reagent, wherein the fixed sample comprises a cross-linked biomolecule of formula (Ia).

[0237]

[0238] Among them, X 1 and X 2The "linker" is the amine-containing moiety of the same or different biomolecules in the sample; the "linker" includes an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12; the uncoupling agent includes a compound capable of cleaving carbamate bonds; optionally, said compound is capable of cleaving carbamate bonds selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof; and

[0239] (b) Detection of analytes from the reaction of the assay reagent and the dissolved biological sample.

[0240] Optionally, the assay method may also include preparing a biological sample by contacting the sample with a fixed reagent composition containing a compound of formula (I) prior to generating discrete partitions (e.g., pores or droplets).

[0241] A wide range of droplet-based assays and systems are known in the art. Assays and systems suitable for use with the compositions and methods of this disclosure include, but are not limited to, the analytical systems described in U.S. Patent Nos. 9,694,361, 1,035,777, 1,027,354, and 1,001,1872, and the analytical systems described in U.S. Patent Application Publications Nos. 20,180105808, 20190367982, and 20190338353, each of which is incorporated herein by reference in its entirety. It is conceivable that any assay that can be performed using a fresh biological sample (e.g., a single cell provided in or encapsulated in a droplet with beads carrying a barcode) can also be performed using a fixed biological sample prepared using a fixation reagent and the relevant methods of this disclosure. That is, in any droplet-based analysis using a fresh biological sample, a droplet-based assay protocol can also be performed, wherein the fresh biological sample is fixed prior to running the assay protocol. In such a assay, the protocol may include providing a fixed biological sample or encapsulating it in discrete droplets, as well as a lysis agent and / or a dissolution agent and assay reagents.

[0242] In some embodiments of the assay method, the discrete partitions (e.g., wells or droplets) further comprise one or more beads. In some embodiments, the beads may contain assay reagents and / or dissolving agents. In some embodiments, a barcode is carried by or contained within the beads. For example, compositions, methods, and systems for preparing, amplifying, and sequencing biomolecules from single-cell samples encapsulated in droplets with barcodes are provided in U.S. Patent Publication No. 20180216162A1, which is incorporated herein by reference.

[0243] Assay reagents may include reagents for performing one or more additional chemical or biochemical operations on biological samples provided or encapsulated in partitions (e.g., in wells, in droplets, or encapsulated in droplets). Therefore, assay reagents usable in this assay method include any reagents that can be used to perform reactions such as nucleic acid modification (e.g., ligation, digestion, methylation, random mutagenesis, bisulfite conversion, uracil hydrolysis, nucleic acid repair, capping or uncapping), nucleic acid amplification (e.g., isothermal amplification or PCR), nucleic acid insertion or cleavage (e.g., CRISPR / Cas9-mediated or transposon-mediated insertion or cleavage), and / or reverse transcription. Additionally, useful assay reagents may include those that allow the preparation of targeted sequences or sequencing reads specific to the target macromolecular component at a higher rate than non-target sequence-specific reads.

[0244] Furthermore, this disclosure provides compositions and systems related to the analysis of fixed biological samples. In one embodiment, this disclosure provides a composition comprising multiple partitions, wherein a subset of said multiple partitions comprises cells fixed using the fixation reagent composition and / or related methods described herein. The partitions may also include lysing agents and / or destabilizing agents. In another embodiment, the partitions of the multiple partitions comprise fixed cells and lysing agents and / or destabilizing agents. In some embodiments, the fixed cells are single fixed cells. In other embodiments, this disclosure provides a composition comprising partitions, wherein the partitions comprise fixed cells prepared using the fixation reagents, lysing agents, and / or destabilizing agents of this disclosure, as described herein. The partitions may be droplets or wells. In another embodiment, the partition may further comprise a protease. In another embodiment, one or more partitions comprising fixed cells as described herein may further comprise an assay reagent, optionally comprising one or more of the following: reverse transcriptase, beads, and reagents for nucleic acid extension reactions. In another embodiment, the compositions of this disclosure are provided at or at a temperature other than ambient temperature. In one embodiment, the temperature is below or above ambient temperature.

[0245] As described elsewhere in this document, partitioning methods can generate a group of partitions or multiple partitions. In such cases, any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions, at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided. Furthermore, the multiple partitions can include unoccupied partitions (e.g., empty partitions) and occupied partitions. For example, the occupancy partition according to this disclosure includes fixed cells (e.g., fixed according to the fixation reagent composition described herein) and a defixing agent.

[0246] On the other hand, this disclosure relates to methods and compositions for dividing a plurality of fixed cells (e.g., fixed according to the fixation reagent composition described herein) into individual partitions. In some cases, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 200, approximately 300, approximately 400, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 2000, approximately 3000, approximately 4000, approximately 5000, approximately 6000, approximately 7000, approximately... Approximately 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 fixed cells can be partitioned into single partitions. In some cases, the method further comprises partitioning approximately 50 to approximately 20,000 fixed cells into partitions using each of a plurality of scaffolds containing a binder (which contains a barcode sequence), wherein the barcode sequence is unique in each of the plurality of scaffolds.

[0247] Figure 10An embodiment of a micropore array is schematically illustrated. This array may be contained within a substrate 1000. The substrate 1000 contains a plurality of pores 1002. The pores 1002 may have any size or shape, and the spacing between the pores, the number of pores per substrate, and the density of pores on the substrate 1000 may vary depending on the specific application. In one such embodiment, a sample molecule 1006, which may contain cells (e.g., fixed or defixed cells) or cellular components (e.g., nucleic acid molecules), is co-located with beads 1004, which may contain nucleic acid barcode molecules coupled thereto. The pores 1002 may be loaded using gravity or other loading techniques (e.g., centrifugation, liquid processors, acoustic loading, photoelectric, etc.). In some cases, at least one of the pores 1002 contains a single sample molecule 1006 (e.g., a cell) and a single bead 1004.

[0248] Reagents can be loaded into the wells sequentially or simultaneously. In some cases, reagents are introduced into the device before or after a specific operation. In others, reagents (which may be provided in the form of droplets or beads in some cases) are introduced sequentially, allowing different reactions or operations to occur at different steps. Reagents (or droplets or beads) can also be loaded in operations in which reaction or operation steps are dispersed. For example, droplets or beads containing reagents for making polynucleotides (e.g., restriction enzymes) and / or other enzymes (e.g., transposases, ligases, polymerases, etc.) can be loaded into one or more wells, followed by droplets or beads containing reagents for attaching nucleic acid barcode molecules to nucleic acid molecules in the sample. Reagents can be provided simultaneously or sequentially with samples, such as cells (e.g., fixed or defixed cells) or cellular components (e.g., organelles, proteins, nucleic acid molecules, carbohydrates, lipids, etc.). Therefore, the use of wells can be useful in multi-step operations or reactions.

[0249] As described elsewhere in this document, nucleic acid barcode molecules and other reagents may be contained within beads or droplets. These beads or droplets may be loaded into partitions (e.g., microwells) before, after, or simultaneously with cell loading (e.g., fixed or unfixed cells), such that each cell is in contact with a different bead or droplet. This technique can be used to attach unique nucleic acid barcode molecules to nucleic acid molecules obtained from each cell (e.g., fixed or unfixed cells). Alternatively or additionally, sample nucleic acid molecules may be attached to a scaffold. For example, a partition (e.g., microwell) may contain beads on which multiple nucleic acid barcode molecules are coupled. Sample nucleic acid molecules or derivatives thereof may be coupled to or attached to nucleic acid barcode molecules on the scaffold. The resulting barcode nucleic acid molecules can then be removed from the partitions, and in some cases, they are collected and sequenced. In such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecules. For example, it can be determined that polynucleotides with the same barcode originate from the same cell or partition, while it can be determined that polynucleotides with different barcodes originate from different cells or partitions.

[0250] Various methods can be used to load samples or reagents into wells or microwells. Samples (e.g., cells or cell components) or reagents (as described herein) can be loaded into wells or microwells using external forces (e.g., gravity, electricity, magnetism) or mechanisms that drive the samples or reagents into the wells (e.g., via pressure-driven flow, centrifugation, photoelectric, acoustic loading, electric pumping, vacuum, capillary flow, etc.). In some cases, fluid handling systems can be used to load samples or reagents into the wells. The loading of samples or reagents can follow a Poisson distribution or a non-Poisson distribution, such as a superpoisson or subpoisson distribution. The geometry of the microwells, the spacing between the wells, the density, and the size can be modified to accommodate a useful distribution of samples or reagents; for example, the size and spacing of the microwells can be adjusted so that the samples or reagents can be distributed in a superpoisson manner.

[0251] In one particular non-limiting embodiment, the microwell array or plate comprises pairs of microwells, wherein each pair of microwells is configured to hold a droplet (e.g., containing a single cell, such as a single fixed cell or a single unfixed cell) and a single bead (as described herein, which may also be provided or encapsulated in the droplet in some cases). The droplet and bead (or droplet containing a bead) may be loaded simultaneously or sequentially, and the droplet and bead may merge, for example, upon contact between the droplet and bead or upon application of a stimulus (e.g., external force, agitation, heat, light, magnetic force, or electricity). In some cases, the loading of the droplet and bead is superpoisonous. In other embodiments of the microwell pair, the wells are configured to hold two droplets containing different reagents and / or samples, which merge upon contact or application of a stimulus. In such cases, the droplet in one microwell of the pair may contain a reagent that can react with a reagent in the droplet of the other microwell of the pair. For example, one droplet may contain a reagent configured to release a nucleic acid barcode molecule contained in a bead in another droplet located in an adjacent microwell. During droplet merging, nucleic acid barcode molecules can be released from the beads into partitions (e.g., contacting micropores or pairs of micropores) and can be further processed (e.g., barcode encoding, nucleic acid reactions, etc.). In the case where cells, such as fixed or decontaminated cells, are loaded in the micropores, one of the droplets can contain reagents for further processing, such as lysing agents for lysing cells during droplet merging.

[0252] Droplets or scaffolds (such as beads) can be partitioned into wells. Droplets can be selected or pretreated before being loaded into the wells. For example, droplets can contain cells, such as fixed or defixed cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for loading into the wells. This pre-selection can be used to efficiently load single cells, such as to obtain a non-Poisson distribution, or to pre-filter cells for selected characteristics before further partitioning in the wells. Additionally, this technique can be used to obtain or prevent the formation of cell duplexes or multiples before or during loading into the microwells.

[0253] In some cases, wells may contain nucleic acid barcode molecules attached thereto. Nucleic acid barcode molecules may be attached to the surface of the well (e.g., the well wall). The nucleic acid barcode molecule in one well (e.g., a partition barcode sequence) may differ from the nucleic acid barcode molecule in another well, which can allow identification of the contents contained in a single partition or well. In some cases, nucleic acid barcode molecules may contain spatial barcode sequences that can identify the spatial coordinates of the well, such as within a well array or plate. In some cases, nucleic acid barcode molecules may contain unique molecular identifiers for the identification of individual molecules. In some cases, nucleic acid barcode molecules may be configured to attach to or capture nucleic acid molecules distributed within a sample or cells (e.g., fixed or unfixed cells) in the well. For example, nucleic acid barcode molecules may contain capture sequences that can be used to capture or hybridize nucleic acid molecules (e.g., RNA, DNA) within the sample. In some cases, nucleic acid barcode molecules may be released from the microwell. For example, nucleic acid barcode molecules may contain chemical crosslinking agents that can be cleaved upon application of a stimulus (e.g., light, magnetism, chemical, biological, etc.). Released nucleic acid barcode molecules (which can be hybridized or configured to hybridize with sample nucleic acid molecules) can be collected and pooled for further processing, which may include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and / or characterization (e.g., sequencing). In such cases, unique partition barcode sequences can be used to identify the cell or partition from which the nucleic acid molecules originated.

[0254] Samples within wells can be characterized. In non-limiting embodiments, such characterization may include imaging of a sample (e.g., cells or cell components) or derivatives thereof. Characterization techniques (such as microscopy or imaging) can be used to measure the profile of a sample in a fixed spatial location. For example, when cells (e.g., fixed or defixed cells) are optionally partitioned with beads, imaging of each well and its contents can provide useful information about cell duplex formation (e.g., frequency, spatial location, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression levels of biomarkers (e.g., surface markers, fluorescently labeled molecules, etc.), cell or bead loading rate, number of cell-bead pairs, and cell-cell interactions (when two or more cells are co-partitioned). Optionally or additionally, imaging may be used to characterize the amount of amplified products in the well.

[0255] During operation, wells can be loaded with samples and reagents simultaneously or sequentially. When cells are loaded (e.g., fixed or defixed cells), the wells can be washed, for example, to remove excess cells from the wells, microwell arrays, or plates. Similarly, washing can be performed to remove excess beads or other reagents from the wells, microwell arrays, or plates. Additionally, cells can be lysed in individual compartments to release intracellular components or analytes. Alternatively, cells can be fixed or permeabilized in individual compartments. Intracellular components or analytes can be coupled to a scaffold, for example, on the surface of a microwell, on a solid scaffold (e.g., beads), or they can be collected for further downstream processing. For example, after cell lysis, intracellular components or analytes can be transferred to individual droplets or other compartments for barcoding. Alternatively or additionally, intracellular components or analytes (e.g., nucleic acid molecules) can be coupled to beads containing nucleic acid barcoded molecules; the beads can then be collected and further processed, for example, by performing nucleic acid reactions such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon can be further characterized, for example, via sequencing. Alternatively or additionally, intracellular components or analytes can be barcoded in the wells (e.g., using beads containing releasable nucleic acid barcoded molecules or on the surface of microwells containing nucleic acid barcoded molecules). The barcoded nucleic acid molecules or analytes can be further processed in the wells, or they can be collected from individual compartments and further processed outside those compartments. Further processing can include nucleic acid processing (e.g., amplification, extension) or characterization (e.g., fluorescence monitoring of amplified molecules, sequencing). At any convenient or useful step, the wells (or microwell arrays or plates) can be sealed (e.g., using oil, membranes, wax, etc.), which allows for the storage of assays or the selective introduction of additional reagents.

[0256] Figure 11 An embodiment workflow for processing nucleic acid molecules within a sample is schematically illustrated. A substrate 1100 comprising a plurality of microwells 1102 may be provided. A sample 1106, which may contain cells (e.g., fixed or deconsolidated cells), cellular components, or analytes (e.g., proteins and / or nucleic acid molecules), may be co-located within the plurality of microwells 1102 with a plurality of beads 1104 containing nucleic acid barcode molecules. During process 1110, the sample 1106 may be processed within the partition. For example, cells may be subjected to conditions sufficient to lyse the cells (e.g., fixed or deconsolidated cells) and release the analytes contained therein. In process 1120, the beads 1104 may be further processed. By way of examples, processes 1120a and 1120b schematically illustrate different workflows depending on the characteristics of the beads 1104.

[0257] In 1120a, the beads contain nucleic acid barcode molecules attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) can be attached to the nucleic acid barcode molecules, for example, via hybridization or ligation. Such ligation can occur on the beads. In process 1130, beads 1104 from multiple wells 1102 can be collected and aggregated. Further processing can be performed in process 1140. For example, one or more nucleic acid reactions, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc., can be performed. In some cases, as described elsewhere herein, a ligation agent sequence is ligated to a nucleic acid molecule or a derivative thereof. For example, sequencing primer sequences can be attached to each end of the nucleic acid molecule. In process 1150, further characterization can be performed, such as sequencing to generate sequencing reads. Sequencing reads can produce information about individual cells or cell populations (e.g., fixed or unfixed cells), which can be represented visually or graphically, for example, in Figure 1155.

[0258] In 1120b, the bead contains a nucleic acid barcode molecule releasably attached thereto, as described below. The bead can be degraded or otherwise release the nucleic acid barcode molecule into well 1102; the nucleic acid barcode molecule can then be used to barcode nucleic acid molecules within well 1102. Further processing can be performed inside or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some cases, as described elsewhere herein, a ligation agent sequence is ligated to the nucleic acid molecule or a derivative thereof. For example, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1150, further characterization can be performed, such as sequencing to generate sequencing reads. Sequencing reads can produce information about individual cells or cell populations (e.g., fixed or unfixed cells), which can be represented visually or graphically, for example, in Figure 1155.

[0259] In 1120b, the bead contains a nucleic acid barcode molecule releasably attached thereto, as described below. The bead can be degraded or otherwise release the nucleic acid barcode molecule into well 1102; the nucleic acid barcode molecule can then be used to barcode nucleic acid molecules within well 1102. Further processing can be performed inside or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some cases, as described elsewhere herein, a ligation agent sequence is ligated to the nucleic acid molecule or a derivative thereof. For example, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1150, further characterization can be performed, such as sequencing to generate sequencing reads. Sequencing reads can produce information about individual cells or cell populations (e.g., fixed or unfixed cells), which can be represented visually or graphically, for example, in Figure 1155.

[0260] I. Other Partition-Based Methods

[0261] This disclosure provides methods and systems for multiplexing and otherwise increasing sample throughput for analysis. For example, single or integrated process workflows can allow for the processing, identification, and / or analysis of more or more analytes, more or more types of analytes, and / or more or more types of analyte characterization. For example, in the methods and systems described herein, one or more markers capable of binding to or otherwise coupling to one or more cells or cell characteristics can be used to characterize cells and / or cell characteristics. In some cases, cell characteristics include cell surface characteristics. Cell surface characteristics may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T-cell receptors, T-cell receptors, B-cell receptors, chimeric antigen receptors, gap junctions, adhesion junctions, or any combination thereof. In some cases, cell characteristics may include intracellular analytes such as proteins, protein modifications (e.g., phosphorylation state or other post-translational modifications), nucleoproteins, nuclear membrane proteins, or any combination thereof. Labelling agents may include, but are not limited to, proteins, peptides, antibodies (or epitope-binding fragments thereof), lipophilic moieties (e.g., cholesterol), cell surface receptor-binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T-cell conjugates, T-cell receptor conjugates, B-cell receptor conjugates, precursors, aptamers, monomers, affinity molecules, darpins, and protein scaffolds, and any combination thereof. Labelling agents may include (e.g., attached to) reporter oligonucleotides indicating the cell surface feature to which the binding group is bound. For example, the reporter oligonucleotide may include a barcode sequence that allows recognition of the labelling agent. For example, a labelling agent specific to one type of cell feature (e.g., a first cell surface feature) may have a first reporter oligonucleotide conjugated thereto, while a labelling agent specific to a different cell feature (e.g., a second cell surface feature) may have different reporter oligonucleotides conjugated thereto. For descriptions of exemplary labelers, reported oligonucleotides, and methods of use, see, for example, U.S. Patent No. 10,550,429; U.S. Patent Publication No. 20190177800; and U.S. Patent Publication No. 20190367969, each of which is incorporated herein by reference in its entirety for all purposes.

[0262] In certain embodiments, a library of potential cell-signature markers can be provided, wherein each cell-signature marker binds to a nucleic acid reporter molecule such that different reporter oligonucleotide sequences bind to each marker capable of binding to a specific cell signature. In other aspects, different members of the library are characterized by the presence of different oligonucleotide sequence markers. For example, an antibody capable of binding to a first protein may have a first reporter oligonucleotide sequence bound to it, while an antibody capable of binding to a second protein may have a different reporter oligonucleotide sequence bound to it. The presence of a specific oligonucleotide sequence may indicate the presence of a specific antibody or a cell signature that can be recognized or bound by a specific antibody.

[0263] For workflows involving the use of fixatives and / or de-fixing agents, labeling agents can be used to label samples (e.g., cells, fixed cells, or de-fixed cells) at different time points. In one embodiment, multiple cells are labeled before and / or after treatment with a fixative. In another embodiment, multiple fixed cells are labeled before and / or after treatment with a de-fixing agent. In yet another embodiment, multiple de-fixed cells are labeled before separating component areas (e.g., wells or droplets) are used for further processing. In yet another embodiment, the methods, compositions, systems, and kits described herein provide labeled cells, labeled fixed cells, or labeled de-fixed cells.

[0264] Labels capable of binding to or otherwise coupling to one or more cells (including fixed and unfixed cells) can be used to characterize cells belonging to a specific cell group. For example, a label can be used to label a cell sample or a group of cells (including fixed and unfixed cells). Thus, one group of cells can be labeled differently from another group of cells. In one embodiment, the first group of cells may be derived from a first sample, and the second group of cells may be derived from a second sample. The label can allow the first and second groups to have different labelers (or reporter oligonucleotides bound to the labelers). This can, for example, facilitate multiplexing, where cells from the first and second groups can be labeled separately and then pooled together for downstream analysis. Downstream detection of the label can indicate that the analyte belongs to a specific group.

[0265] For example, a reporter oligonucleotide may be linked to an antibody or its epitope-binding fragment, and labeled cells (including fixed or defixed cells) may include barcode molecules that enable antibody-linked or epitope-binding fragment-linked molecules to meet conditions suitable for binding the antibody to molecules present on the cell surface. The binding affinity between the antibody or its epitope-binding fragment and the molecules present on the surface is within a desired range to ensure that the antibody or its epitope-binding fragment remains bound to the molecules. For example, the binding affinity is within a desired range to ensure that the antibody or its epitope-binding fragment remains bound to the molecules during various sample processing steps, such as partitioning and / or nucleic acid amplification or extension. The dissociation constant (Kd) between an antibody or its epitope-binding fragment and the molecule it binds to can be less than approximately 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 5 0 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM. For example, the dissociation constant can be less than about 10 μM.

[0266] In another embodiment, the reporter oligonucleotide may be coupled to a cell-penetrating peptide (CPP), and the labeled cell may comprise the delivery of the CPP-conjugated reporter oligonucleotide into an analyte carrier. The labeled analyte carrier may comprise the delivery of the CPP-conjugated oligonucleotide to cells and / or cell beads via the cell-penetrating peptide. The CPP used in the methods provided herein may contain at least one nonfunctional cysteine ​​residue, which may be free or derived to form a disulfide bond with an oligonucleotide modified for such linkage. Non-limiting examples of CPPs that may be used in the embodiments described herein include permein, transporter proteins, plsl, TAT (48-60), pVEC, MTS, and MAP. The cell-penetrating peptide used in the methods provided herein may have the ability to induce at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% cell penetration of a cell population. The CPP may be an arginine-rich peptide transporter protein. CPPs can be penetratin or Tat peptides. In another embodiment, the reported oligonucleotide can be coupled to a fluorophore or dye, and the labeled cell may contain a barcode molecule that links the fluorophore, subjecting it to conditions suitable for binding the fluorophore to the cell surface. In some cases, the fluorophore may interact strongly with the lipid bilayer, and the labeled cell may contain a barcode molecule that links the fluorophore, subjecting it to conditions that allow the fluorophore to bind to or insert into the cell membrane. In some cases, the fluorophore is a water-soluble organic fluorophore. In some cases, the fluorophore is Alexa532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, sulfonyl-Cy3 maleimide, Alexa546 carboxylic acid / succinimide ester, Atto550 maleimide, Cy3 carboxylic acid / succinimide ester, Cy3B carboxylic acid / succinimide ester, Atto565 biotin, sulfonylrhodamine B, Alexa594 maleimide, Texas red maleimide, Alexa633 maleimide, AbberiorSTAR635P azide, Atto647N maleimide, Atto647SE, or sulfonyl-Cy5 maleimide. See, for example, Hughes LD et al., PLOS ONE, Feb 4, 2014; 9(2):e87649, which describes organic fluorophores and is incorporated herein by reference in its entirety for all purposes.

[0267] Reporter oligonucleotides can be coupled to lipophilic molecules, and labeled cells can contain the delivery of nucleic acid barcode molecules to the cell membrane or nuclear membrane via lipophilic molecules. Lipophilic molecules can bind to and / or insert into lipid membranes (such as cell and nuclear membranes). In some cases, the insertion can be reversible. In some cases, the binding between the lipophilic molecule and the cell or nuclear membrane allows the membrane to retain the lipophilic molecule (e.g., and its bound components, such as nucleic acid barcode molecules) during subsequent processing (e.g., partitioning, cell permeabilization, amplification, pooling, etc.). Reporter oligonucleotides can enter the intracellular space and / or the cell nucleus. In one embodiment, the reporter oligonucleotide coupled to the lipophilic molecule will remain bound to and / or inserted into the lipid membrane (as described herein) via the lipophilic molecule until cell lysis occurs, for example, within a partition.

[0268] Reported oligonucleotides can be part of a nucleic acid molecule that contains any number of functional sequences, such as targeting capture sequences, random primer sequences, etc., as described elsewhere in this document, and is coupled to another nucleic acid molecule that is an analyte or derived from an analyte.

[0269] Prior to partitioning, cells can be incubated with a library of markers, which can be markers targeting a broad set of different cellular characteristics (e.g., receptors, proteins, etc.) and include their associated reporter oligonucleotides. Unbound markers can be washed from the cells, and the cells can then be co-separated (e.g., separated into droplets or pores) with partition-specific barcode oligonucleotides (e.g., attached to a scaffold, such as beads or gel beads), as described elsewhere herein. Thus, partitioning can include cells or one or more cells, along with bound markers and their known associated reporter oligonucleotides.

[0270] In other cases, for example, to facilitate sample multiplexing, cell-specific markers may have a first plurality of markers (e.g., antibodies or lipophilic moieties) coupled to a first reporter oligonucleotide and a second plurality of markers coupled to a second reporter oligonucleotide. For example, the first plurality of markers and the second plurality of markers may interact with different cells, cell populations, or samples, allowing a specific reporter oligonucleotide to indicate a specific cell population (or cell or sample) and cell characteristic. In this way, different samples or groups can be processed independently and subsequently combined for pooled analysis (e.g., partition-based barcode encoding as described elsewhere herein). See, for example, U.S. Patent Publication No. 20190323088, which is incorporated herein by reference in its entirety for all purposes.

[0271] As described elsewhere in this document, libraries of markers can be combined with specific cellular characteristics and analytes used to identify cells derived from a particular cell population or sample. Cell populations can be incubated with multiple libraries, resulting in cells or one or more cells containing multiple markers. For example, cells can contain lipophilic markers and antibodies conjugated thereto. Lipophilic markers indicate that the cells are members of a specific cell sample, while antibodies indicate that the cells contain a specific analyte. In this way, reported oligonucleotides and markers can allow for multi-analyte, multiplexed analysis.

[0272] In some cases, these reporter oligonucleotides can contain nucleic acid barcode sequences that allow for the identification of markers coupled to the reporter oligonucleotide. Using oligonucleotides as reporter molecules offers the advantage of generating significant diversity in sequence, while also allowing for easy attachment to most biomolecules, such as antibodies, and easy detection, for example, using sequencing or array techniques.

[0273] The attachment (coupling) of oligonucleotides to labeling agents can be achieved through a variety of direct or indirect, covalent or non-covalent binding or attachment methods. For example, chemical conjugation techniques can be used (e.g., available from Anolon Biotechnology Co., Ltd.). Antibody labeling kits and other non-covalent attachment mechanisms, such as using biotinylated antibodies and oligonucleotides with anti-biotin or streptavidin linkers (or beads comprising one or more biotinylated linkers coupled to the oligonucleotides), covalently attach oligonucleotides to a portion of a labeling agent (such as a protein, such as an antibody or antibody fragment). Antibody and oligonucleotide biotinylation techniques are available. See, for example, Fang et al., “5′-terminal labeling and affinity purification of fluoride-cleavable biotinylated phosphoramidites for the synthesis of oligonucleotides,” Journal of Nucleic Acid Research, January 15, 2003; 31(2): 708-715, which is incorporated herein by reference in its entirety for all purposes. Similarly, protein and peptide biotinylation techniques have been developed and are readily available. See, for example, U.S. Patent No. 6,265,552, which is incorporated herein by reference in its entirety for all purposes. In addition, click reaction chemistry, such as the methyltetraazine-PEG5-NHS ester reaction and the TCO-PEG4-NHS ester reaction, can be used to conjugate reporter oligonucleotides to labeling agents. Commercially available kits, such as those from Thunderlink and Abcam, as well as techniques commonly used in the art, can be used to suitably conjugate reporter oligonucleotides to labeling agents. In another embodiment, the labeling agent is indirectly (e.g., via hybridization) conjugated to a reporter oligonucleotide containing a barcode sequence that recognizes the labeling agent. For example, the labeling agent can be directly conjugated (e.g., covalently bound) to a hybrid oligonucleotide containing a sequence that hybridizes with the reporter oligonucleotide sequence. Hybridization of the hybrid oligonucleotide to the reporter oligonucleotide conjugates the labeling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotide is releasable from the labeling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide can be attached to the labeling agent via unstable bonds (e.g., chemically unstable, photosensitive, thermally unstable, etc.), as generally described elsewhere herein for releasing molecules from a scaffold. In some cases, the reported oligonucleotides described herein may include one or more functional sequences that can be used for subsequent processing, such as ligand sequences, unique molecular identifier (UMI) sequences, sequencer-specific flow cell attachment sequences (such as P5, P7, or partial P5 or P7 sequences), primers or primer-binding sequences, sequencing primers or primer-binding sequences (such as R1, R2, or partial R1 or R2 sequences).

[0274] In some cases, the labeling agent may comprise a reporter oligonucleotide and a label. The label may be a fluorophore, a radioisotope, a molecule capable of colorimetric reactions, a magnetic particle, or any other suitable molecule or compound capable of detection. The label may be conjugated directly or indirectly to the labeling agent (or reporter oligonucleotide) (e.g., the label may be conjugated to a molecule capable of binding to the labeling agent or reporter oligonucleotide). In some cases, the label is conjugated to an oligonucleotide whose sequence is complementary to the reporter oligonucleotide, and hybridization between the oligonucleotide and the reporter oligonucleotide may be permitted.

[0275] Figure 12 Exemplary labelers (1210, 1220, 1230) comprising a reporter oligonucleotide (1240) attached thereto are described. Labeler 1210 (e.g., any labeler described herein) is attached (directly, e.g., covalently, or indirectly) to reporter oligonucleotide 1240. Reporter oligonucleotide 1240 may contain a barcode sequence 1242 that identifies labeler 1210. Reporter oligonucleotide 1240 may also contain one or more functional sequences 1243 that can be used for subsequent processing, such as a ligator sequence, a unique molecular identifier (UMI) sequence, a sequencer-specific flow cell attachment sequence (e.g., P5, P7, or a portion of P5 or P7 sequence), a primer or primer-binding sequence, or a sequencing primer or primer-binding sequence (e.g., R1, R2, or a portion of R1 or R2 sequence).

[0276] refer to Figure 12 In some cases, the reporter oligonucleotide 1240 conjugated to a labeler (e.g., 1210, 1220, 1230) comprises a primer sequence 1241, a barcode sequence 1242 recognizing the labeler (e.g., 1210, 1220, 1230), and a functional sequence 1243. The functional sequence 1243 can be configured to hybridize with a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule 1290 (not shown), as described elsewhere herein. In some cases, the nucleic acid barcode molecule 1290 is attached to a scaffold (e.g., beads, such as gel beads), as described elsewhere herein. For example, the nucleic acid barcode molecule 1290 can be attached to a scaffold via a releasable bond (e.g., containing an unstable bond), as described elsewhere herein. In some cases, the reporter oligonucleotide 1240 comprises one or more additional functional sequences, as described above.

[0277] In some cases, labeler 1210 is a protein or polypeptide (e.g., an antigen or intended antigen) comprising reporter oligonucleotide 1240. Reporter oligonucleotide 1240 comprises a barcode sequence 1242 that recognizes polypeptide 1210 and can be used to infer the presence of an analyte, such as a binding partner of polypeptide 1210 (i.e., a molecule or compound that polypeptide 1210 can bind). In some cases, labeler 1210 is a lipophilic moiety (e.g., cholesterol) comprising reporter oligonucleotide 1240, wherein the lipophilic moiety is selected such that labeler 1210 integrates into the cell membrane or nuclear membrane. Reporter oligonucleotide 1240 comprises a barcode sequence 1242 that recognizes the lipophilic moiety 1210, which in some cases is used to label cells (e.g., cell groups, cell samples, etc.) and can be used for multiplexing analyses as described elsewhere herein. In some cases, labeler is an antibody 1220 (or an epitope-binding fragment thereof) comprising reporter oligonucleotide 1240. The reporter oligonucleotide 1240 contains a barcode sequence 1242 that recognizes antibody 1220 and can be used to infer the presence of, for example, a target of antibody 1220 (i.e., a molecule or compound that antibody 1220 binds to). In other embodiments, the labeler 1230 contains an MHC molecule 1231 that includes a peptide 1232 and a reporter oligonucleotide 1240 that recognizes peptide 1232. In some cases, the MHC molecule is coupled to a scaffold 1233. In some cases, the scaffold 1233 may be a polypeptide, such as streptavidin, or a polysaccharide, such as dextran. In some cases, the reporter oligonucleotide 1240 may be coupled directly or indirectly to the MHC labeler 1230 in any suitable manner. For example, the reporter oligonucleotide 1240 may be coupled to MHC molecule 1231, scaffold 1233, or peptide 1232. In some embodiments, the labeler 1230 contains multiple MHC molecules (e.g., MHC multimers that can be coupled to a scaffold (e.g., 1233)). Many possible configurations of class I and / or class II MHC polymers can be used with the compositions, methods, and systems disclosed herein, such as MHC tetramers, MHC pentamers (MHCs assembled via coiled-helix domains, e.g.) MHC pentamers (ProImmune Ltd.), MHC octamers, MHC dodecamers, and MHC-modified dextran molecules (e.g.) (Immudex), etc. For descriptions of exemplary markers, including antibodies and MHC-based markers, reported oligonucleotides, and methods of use, see, for example, U.S. Patent No. 10,550,429 and U.S. Patent Publication No. 20190367969, each of which is incorporated herein by reference in its entirety for all purposes.

[0278] Figure 13Another embodiment of beads carrying barcodes is illustrated. In some embodiments, analysis of multiple analytes (e.g., RNA using the labeling agents described herein and one or more analytes) may include nucleic acid barcode molecules, such as... Figure 13 The scaffold is generally described herein. In some embodiments, nucleic acid barcode molecules 1310 and 1320 are attached to the scaffold 1330 via releasable bonds 1340 (e.g., containing unstable bonds) as described elsewhere herein. Nucleic acid barcode molecule 1310 may comprise a linker sequence 1311, a barcode sequence 1312, and a linker sequence 1313. Nucleic acid barcode molecule 1320 may comprise a linker sequence 1321, a barcode sequence 1312, and a linker sequence 1323, wherein the linker sequence 1323 comprises a sequence different from the linker sequence 1313. In some cases, linker 1311 and linker 1321 comprise the same sequence. In some cases, linker 1311 and linker 1321 comprise different sequences. Although the scaffold 1330 is shown as comprising nucleic acid barcode molecules 1310 and 1320, any suitable number of barcode molecules comprising a common barcode sequence 1312 are contemplated herein. For example, in some embodiments, the scaffold 1330 further includes a nucleic acid barcode molecule 1350. The nucleic acid barcode molecule 1350 may include a linker sequence 1351, a barcode sequence 1312, and a linker sequence 1353, wherein the linker sequence 1353 includes a sequence different from the linker sequences 1313 and 1323. In some cases, the nucleic acid barcode molecule (e.g., 1310, 1320, 1350) includes one or more additional functional sequences, such as UMI or other sequences described herein. The nucleic acid barcode molecules 1310, 1320, or 1350 may interact with analytes described elsewhere herein, for example, such as... Figure 14A -C describes it.

[0279] refer to Figure 14AIn the case of labeling cells with a marker, sequence 1423 may be complementary to the linker sequence of the reporter oligonucleotide. Cells may be contacted with a marker 1410 (e.g., a peptide, antibody, or others described elsewhere herein) conjugated to one or more reporter oligonucleotides 1510. In some cases, cells may be further processed prior to barcode encoding. For example, such processing steps may include one or more washing and / or cell sorting steps. In some cases, cells bound to the marker 1410 (which is conjugated to oligonucleotide 1510 and a scaffold 1430 containing a nucleic acid barcode molecule 1490 (e.g., beads, such as gel beads)) are separated into partitions within multiple zones (e.g., droplets of a droplet emulsion or pores of a micropore array). In some cases, a partition may contain at most a single cell bound to the marker 1410. In some cases, the reporting oligonucleotide 1510 conjugated to labeler 1410 (e.g., peptides, antibodies, pMHC molecules, such as MHC multimers, etc.) comprises a first linker sequence 1411 (e.g., a primer sequence), a barcode sequence 1412 recognizing labeler 1410 (e.g., a peptide, antibody, or peptide that recognizes a pMHC molecule or complex), and a linker sequence 1413. Linker sequence 1413 may be configured to hybridize with a complementary sequence, such as sequence 1423 present on nucleic acid barcode molecule 1490. In some cases, oligonucleotide 1510 comprises one or more additional functional sequences, as described elsewhere herein.

[0280] Barcode-encoded nucleic acids can be obtained from Figure 14A The construct described in C is generated (e.g., via nucleic acid reactions such as nucleic acid extension or ligation). For example, sequence 1413 can then be hybridized with complementary sequence 1423 to generate (e.g., via nucleic acid reactions such as nucleic acid extension or ligation) a barcoded nucleic acid molecule containing cell (e.g., partition-specific) barcode sequence 1422 (or its reverse complementary sequence) and reporter sequence 1412 (or its reverse complementary sequence). The barcode-encoded nucleic acid molecule can then optionally be processed as described elsewhere herein, for example, by amplifying the molecule and / or attaching sequencing platform-specific sequences to the fragment. See, for example, U.S. Patent Publication No. 2018 / 0105808, which is incorporated herein by reference in its entirety for all purposes. The barcode-encoded nucleic acid molecule or derivatives thereof can then be sequenced on a suitable sequencing platform.

[0281] In some cases, analysis of multiple analytes (e.g., nucleic acids using the labeling agents described herein and one or more analytes) can be performed. For example, the workflow may include, for instance, Figure 14A -C Any described workflow or combination of workflows for a single analyte, as described elsewhere in this document. For example, by using a combination of workflows, such as Figure 14AAs described by -C, it can analyze a variety of analytes.

[0282] In some cases, the analysis of analytes (e.g., nucleic acids, peptides, carbohydrates, lipids, etc.) includes, for example, Figure 14A The workflow is described in general. Nucleic acid barcode molecule 1490 may co-occur with one or more analytes. In some cases, nucleic acid barcode molecule 1490 is attached to scaffold 1430 (e.g., beads, such as gel beads), as described elsewhere herein. For example, nucleic acid barcode molecule 1490 may be attached to scaffold 1430 via releasable bonds 1440 (e.g., containing unstable bonds), as described elsewhere herein. Nucleic acid barcode molecule 1490 may contain barcode sequence 1421 and optionally other additional sequences, such as UMI sequence 1422 (or other functional sequences described elsewhere herein). Nucleic acid barcode molecule 1490 may contain sequence 1423 that is complementary to another nucleic acid sequence, allowing it to hybridize with a specific sequence.

[0283] For example, sequence 1423 may contain a poly-T sequence and can be used for hybridization with mRNA. (Reference) Figure 14C In some embodiments, the nucleic acid barcode molecule 1490 includes a sequence 1423 complementary to the sequence of the RNA molecule 1460 from the cell. In some cases, sequence 1423 includes a sequence specific to the RNA molecule. Sequence 1423 may include a known or targeted sequence or a random sequence. In some cases, a nucleic acid extension reaction may be performed to generate a barcode-encoded nucleic acid product comprising sequence 1423, barcode sequence 1421, UMI sequence 1422, any other functional sequences, and a sequence corresponding to the RNA molecule 1460.

[0284] In another embodiment, sequence 1423 may be complementary to a protruding end sequence or a linker sequence already attached to the analyte. For example, refer to Figure 14BIn some embodiments, primer 1450 contains a sequence complementary to a sequence of nucleic acid molecule 1460 from an analyte vector (such as RNA encoding a BCR sequence). In some cases, primer 1450 contains one or more sequences 1451 that are not complementary to RNA molecule 1460. Sequence 1451 may be a functional sequence as described elsewhere herein, such as a ligation sequence, a sequencing primer sequence, or a sequence that facilitates coupling to a sequencer in flowing cells. In some cases, primer 1450 contains a poly-T sequence. In some cases, primer 1450 contains a sequence complementary to a target sequence in the RNA molecule. In some cases, primer 1450 contains a sequence complementary to a region of an immune molecule (such as a constant region of a TCR or BCR sequence). Primer 1450 hybridizes with nucleic acid molecule 1460 and generates a complementary molecule 1470. For example, complementary molecule 1470 may be cDNA generated in a reverse transcription reaction. In some cases, additional sequences are attached to complementary molecule 1470. For example, a reverse transcriptase can be selected to attach several untemplated bases 1480 (e.g., a poly-C sequence) to cDNA. In another embodiment, a terminal transferase can also be used to attach additional sequences. Nucleic acid barcode molecule 1490 contains a sequence 1424 complementary to the untemplated bases, and a reverse transcriptase performs a template-switching reaction on nucleic acid barcode molecule 1490 to generate a barcode-encoded nucleic acid molecule containing a cell (e.g., region-specific) barcode sequence 1422 (or its reverse complementary sequence) and a sequence (or a portion thereof) of complementary molecule 1470. In some cases, sequence 1423 contains a sequence complementary to a region of an immune molecule (such as a constant region of a TCR or BCR sequence). Sequence 1423 hybridizes with nucleic acid molecule 1460, generating complementary molecule 1470. For example, complementary molecule 1470 can be generated in a reverse transcription reaction that produces a barcoded nucleic acid molecule containing a cell (e.g., partition-specific) barcoded sequence 1422 (or its reverse complementary sequence) and a sequence (or a portion thereof) of complementary molecule 1470. Methods and compositions (including template-converting oligonucleotides) suitable for barcoding and / or barcoding cDNA generated from mRNA transcripts (including those encoding the V(D)J region of immune cell receptors) are described in International Patent Application WO2018 / 075693, U.S. Patent Publication No. 2018 / 0105808, U.S. Patent Publication No. 2015 / 0376609, and U.S. Patent Publication No. 2019 / 0367969, filed June 26, 2015, each of which is incorporated herein by reference in its entirety for all purposes.

[0285] Example

[0286] Various features and embodiments of this disclosure are illustrated in the following representative examples, which are intended to be illustrative and not restrictive. Those skilled in the art will readily understand that the specific examples are merely illustrative of embodiments of this disclosure, as more fully described in the following claims. Each embodiment and feature described herein should be understood to be interchangeable and combined with each embodiment contained therein.

[0287] Example 1: Synthesis of a reversible immobilization reagent

[0288] This embodiment illustrates the synthesis and use of the reversible fixation reagent ethane-1,2-diacylbis(1H-imidazol-1-carboxylic acid) (compound (2a)). The fixation reagent of compound (2a) reacts with the amine-containing portion of biomolecules in a biological sample to form a dicarbamate crosslink, which is reversibly treated with a de-fixing agent (e.g., DETA) that cleaves the carbamate bonds.

[0289] Synthesis of compound (2a): The synthesis of compound (2a) is summarized in scheme 7.

[0290] Option 7

[0291]

[0292] All reagents were commercially available (Merck Sigma) and could be used without further purification. 20 g of carbonyl diisoimidazole (“CDI”) (123 mmol, 3.0 equiv) was added to a 250 mL single-necked RBF containing a stir bar. 100 mL of dichloromethane (“DCM”) solvent was added, and the solution was rapidly stirred to obtain a fine white suspension. Glycerol (2.30 mL, 40.1 mmol, 1 equiv) was added via syringe. The reaction vessel was sealed and covered with an argon balloon to maintain an inert atmosphere. The reaction was allowed to proceed for 16 hours, during which the solution changed from a white suspension to a clear pale yellow. The resulting solution was washed three times with 100 mL of deionized water and dried over MgSO4. The solvent was removed under vacuum to provide a fine white / off-white powder. The formation of the desired product of compound (2a) was confirmed by NMR, showing three peaks between 7.0 and 8.5 ppm and singlets of two CH2 groups with alkyl bridges. As described in Example 2, the resulting compound (2a) was used as a fixative without further purification.

[0293] Synthesis of compound (5a): The synthesis of bis-imidazolium-glycerol (compound (5a)) is summarized in scheme 8.

[0294] Option 8

[0295]

[0296] The bis-imidazolium glycerol scaffold for compound (5a) was synthesized under the conditions used for compound (2a), but with the addition of 2.5 equivalents of CDI in dichloromethane, and the reaction was allowed to proceed at room temperature for 16 hours. The formation of compound (5a) can be achieved by... 1 Identification was performed by ¹H NMR, which involved integrating the aryl peak (δ = 7.00-8.5 ppm) and the CH2 peak (δ = 4.6 ppm) corresponding to imidazole (δ = 7.00-8.5 ppm) at 6H and 4H, respectively.

[0297] Synthesis of compound (5b): The synthesis of acetyl-bis-imidazolium-glycerol (compound (5b)) is summarized in scheme 9.

[0298] Option 9

[0299]

[0300] Compound (5b) is an acetylated analog of compound (5a), synthesized under the conditions described for compound (2a) above, but with the addition of 1.5 equivalents of acetic anhydride after 16 hours, and the reaction was allowed to be stirred for another 4 hours. The acetylated product was identified by the presence of a CH3 peak at δ = 2.00 ppm and a multi-peak broadening corresponding to the glycerol backbone.

[0301] Synthesis of compound (5c): The synthesis of tri-imidazolium-glycerol (compound (5c)) is summarized in scheme 10.

[0302] Option 10

[0303]

[0304] Compound (5c) was synthesized under the conditions described for compound (2a), but with the excess CDI in dichloromethane increased fourfold. The reaction was carried out at room temperature for 16 hours. The product was confirmed to be the result of the integration of nine aromatic protons and five alkyl protons.

[0305] Example 2: Use of reversible fixation reagent

[0306] This example illustrates the use of a reversible fixation reagent compound (2a). The fixation reagent of compound (2a) reacts with the amine-containing portion of biomolecules in a biological sample to form a dicarbamate crosslink, which is reversibly removed by treatment with a defixing agent that cleaves the carbamate bonds (e.g., DETA). This example illustrates the use of compound (2a) to fix PBMCs, followed by storage for up to 21 days, and then defixation with DETA to produce RNA.

[0307] PBMC Immobilization: Compound (2a) prepared as described in Example 1 was prepared as a 200 mM stock solution in anhydrous DMSO. The final targeted immobilizer was prepared by diluting the stock solution in PBS to provide the desired concentration. The prepared compound (2a) immobilizer was used within 10 minutes after preparation to prevent hydrolysis by reactive imidazole-carboxylate salts. PBMCs were immobilized in 100 μL of 15 mM compound (2a) immobilizer for 30 minutes or 1 hour, and then quenched in PBS with 10% fetal bovine serum (FBS). For comparison, individual PBMC samples were immobilized in 1 mL of 4% paraformaldehyde (“PFA”) in PBS for 20 minutes, and then quenched with 10% FBS in PBS. The immobilized PBMCs were stored at room temperature, 4°C, or -80°C for 21 days.

[0308] RNA assay of decontaminated PBMCs: At weekly time points, cells from peripheral blood mononuclear cell samples fixed with PBMCs were centrifuged at 450 g for 5 min, and the supernatant was collected and retained. Decontaminated (or “decrosslinked”) PBMC particles fixed with compound (2a) were incubated for 15 min in 0.1% diethylenetriamine (DETA) solution in PBS at 50 °C or for 2 h in 0.1% sodium dodecyl sulfate in PBS / 0.3% foscholine in PBS at 40 °C. PFA-fixed PBMC particles were decontaminated by incubation at 0.1% sodium dodecyl sulfate (SDS), 30 mM Tris, pH 6.8, 40 °C for 2 h. After decontamination, PBMCs were centrifuged at 450 g for 5 min, and particles (if any) and supernatant were collected.

[0309] RNA was isolated from the supernatant obtained before treatment with the activator (labeled "Storage Solution"), and the supernatant collected after treatment with the deactivator (labeled "Supernatant") was separated using the RNAeasy MinElute Cleaning Kit (Kiagen, Catalog No. 74204). RNA was then isolated from the cell particles ("Particles") obtained after treatment with the deactivator using the RNAeasy Plus Mini Kit (Kiagen, Catalog No. 74134). The isolated RNA was obtained using qubits. TM The RNA-HS assay kit (Ingenie, catalog number Q32855) and the Agilent RNA screening tape system (Agilent Technologies) were used for evaluation.

[0310] Results: As shown in Table 3, all fresh or fixative-treated samples exhibited significant RNA leakage into the storage solution during storage. Samples treated with fixative for 60 minutes and stored for 21 days showed significantly higher levels of RNA recovered from particles and supernatant even after 21 days of storage, with relatively low leakage.

[0311] Table 3

[0312]

[0313] Example 3: Solvent conditions using reversible fixation reagent

[0314] The results of Example 2 indicate that some RNA leakage occurred in peripheral blood mononuclear cells fixed with compound (2a), and it is hypothesized that the solvent conditions used for fixation, particularly the amount of DMSO present, may have contributed to this leakage. This example illustrates the experimental study that determined the optimal solvent conditions for using the reversible fixation reagent compound (2a) to fix PBMC biological samples.

[0315] Materials and Methods: Compound (2a) was prepared as described in Example 1 and prepared as a 200 mM stock solution in anhydrous DMSO. Fixative solutions of compound (2a) were prepared by diluting the stock solution in PBS at the following fixative reagents and DMSO concentrations: 15 mM compound (2a) in 15% DMSO; 15 mM compound (2a) in 10% DMSO; 15 mM compound (2a) in 7.5% DMSO; 10 mM compound (2a) in 5% DMSO; or 5 mM compound (2a) in 2.5% DMSO. Fresh PBMCs were fixed in 100 μL of each fixative for 30 min or 1 h, quenched with 100 mM Tris, and then washed in 10% FBS in PBS.

[0316] At day 0 and day 3, cells from fixed PBMC samples were centrifuged at 450 g for 5 min, and the supernatant was collected and retained. Particles of cells fixed with compound (2a) were destabilized by incubation in 1% diethylenetriamine (DETA) solution. After destabilization treatment, samples were centrifuged at 450 g for 5 min, and particles (if present) and supernatant were collected. RNA was isolated from the supernatant obtained before destabilization treatment (labeled “Store Solution”), and the supernatant collected after destabilization treatment (labeled “Supernatant”) was separated using the RNAeasy MinElute Clean Kit (Kiagen, Catalog No. 74204). RNA was isolated from cell particles (“Particles”) obtained after destabilization treatment using the RNAeasy Plus Mini Kit (Kiagen, Catalog No. 74134). The isolated RNA was subjected to quality recovery and quality assessment (using qubits or Tapestation, respectively).

[0317] Results: It was observed that decreasing DMSO concentration during fixation with compound (2a) resulted in increased RNA retention. For example, cells in samples fixed with a 2.5% DMSO solution containing only 5 mM of compound (2a) showed significantly higher RNA retention after 3 days at 4C compared to solutions of compound (2a) fixed with 15% DMSO in 10% or 15% DMSO.

[0318] Example 4: Preparation of reversibly immobilized biological samples in discrete droplets using carbamate reversible desolvation agents

[0319] This embodiment illustrates the preparation of discrete droplets (GEMs) containing a biological sample of PBMCs previously immobilized with compound (2a) and the carbamate cleavage and destabilizing agent DETA, and then using the destabilized sample in the droplets to perform single-cell RNA sequence expression profiling experiments.

[0320] Preparation of immobilized biological samples:

[0321] Fixed biological samples of immobilized PBMCs were prepared as described in Example 1 above. The immobilized biological samples could be stored at 4C or -20C for several days or longer before being processed in droplet-based assays (e.g., single-cell assays).

[0322] Preparation of the anti-solidifying agent:

[0323] As described in Example 2, a 0.1% DETA stock solution was prepared.

[0324] Droplet generation (GEM) was performed using fixed cells, a deconsolidating agent, and barcode gel beads.

[0325] A fixed biological sample containing immobilized PBMCs is converted into a standard master mixture for use with the Chromium system (10x Genomics, Pleasanton, CA, USA) to partition the sample together with barcode-encoded gel beads (“gel beads in emulsion”) in the form of discrete droplets called GEMs. The Chromium system is prepared using a dissolving agent solution added as a separate reagent when generating GEMs containing sample PBMCs and barcode gel beads. Alternatively, the dissolving agent solution is added to a reservoir containing a suspension of barcode gel beads and introduced into the GEMs through the same inlet channel as the gel beads. Once generated, the GEMs are collected and a heat incubation step is performed. The heating step facilitates the cleavage and release of cellular contents, barcode oligonucleotides, and the RT reaction leading to cDNA synthesis (incorporating the barcode into the 3' synthon). When the dissolving agent is combined with the GEMs, the heat incubation step can be extended as needed to allow for the deblocking reaction of DETA, which removes carbamate crosslinks from the biomolecules released from the PBMC sample in the GEM.

[0326] Example 5: Reversible fixation using compound (2a) and the enzymatic fixative carboxylesterase (CES)

[0327] This example illustrates the use of compound (2a) to fix Jurkats cells, which are then stored for up to 3 days and defixed using a carboxylesterase to produce RNA.

[0328] Materials and methods:

[0329] A. Preparation of the stock solution:

[0330] The fixative for compound (2a) was prepared as described in Example 1. A 200 mM stock solution of compound (2a) was prepared in anhydrous DMSO.

[0331] Stock solutions of the small molecule destabilizing agents EDA, hydrazine, forscolin, glycine, and ethanolamine were prepared as follows. EDA, hydrazine, or ethanolamine was diluted in 50 mM Tris buffer at pH 8.3 to prepare a 0.1% w / v solution. Glycine was diluted in 50 mM Tris buffer at pH 8.3 to prepare a 100 mM solution. In some groups, forscolin was added to provide a 2% final concentration. To each prepared solution, a final concentration of 1 unit / μL of the Qiager RNase inhibitor (Qiager, catalog number 129916) was added.

[0332] Stock solutions of the enzymatic hydrolase carboxylesterase (“CES”) were prepared as follows. Carboxylesterase 1 or 2 was diluted in 50 mM Tris buffer at pH 8.3 to a final concentration of 1 unit / μL. In some groups, forosclin was added to provide a final concentration of 2%. To each prepared solution, a final concentration of 1 unit / μL of Qiager RNase inhibitor (Qiager, catalog number 129916) was added.

[0333] B. Jurkats' fixation:

[0334] The final targeting fixative was prepared by diluting the stock solution in PBS to provide a 10 mM concentration. The prepared compound (2a) fixative was used within 10 minutes of preparation to prevent reactive imidazole-carboxylate hydrolysis. Fresh Jurkats cells were fixed in 100 μL of 10 mM compound (2a) fixative for 1 hour and then quenched with 10% fetal bovine serum (FBS) in PBS.

[0335] C. Treatment with a cell-fixing agent:

[0336] At day 0 and day 3, cells from the fixed samples were centrifuged at 300g for 5 minutes, and the supernatant was collected and retained. Particles of cells fixed with compound (2a) were destabilized by incubation with a carboxylesterase or a small molecule destabilizing agent, as follows: Cell particles were resuspended and incubated for 15 minutes at 50°C with a destabilizing solution containing or without forosclin-containing EDA, hydrazine, ethanolamine, or hydrazine. Cell particles were also resuspended and incubated for 30 minutes at 37°C with a destabilizing solution containing or without forosclin-containing CES 1 or 2.

[0337] D. RNA extraction and quantification

[0338] After treatment with the deconsolidating agent, the sample was centrifuged at 300g for 5 minutes, and particles (if any) and supernatant were collected. RNA was isolated from the supernatant obtained before treatment with the deconsolidating agent (labeled "Store Solution"), and the supernatant collected after deconsolidation agent treatment (labeled "Supernatant") was separated using the RNAeasyMinElute Cleanup Kit (Kiagener, Catalog No. 74204). RNA was isolated from the cell particles ("Particles") obtained after deconsolidation agent treatment using the RNAeasyPlus Mini Kit (Kiagener, Catalog No. 74134). The isolated RNA was subjected to quality recovery and quality assessment (using qubits or Tapestation, respectively).

[0339] Results: As shown in Table 4, the use of the enzymatic decongestant carboxylesterase resulted in a higher RNA recovery rate (relative to fresh cells) from cells fixed with compound (2a) compared to other small molecule decongestants such as EDA, hydrazine, forscolin, glycine, and ethanolamine. In fact, the total RNA recovered was comparable to the amount recovered from decongested fresh cells.

[0340] Table 4

[0341] sample Decontaminant treatment Total recovered RNA (ng) Fresh 1237.5 Fixed none 84.2 Fixed 0.1% EDA 147.2 Fixed 0.1% EDA + 2% Forscorin 97.7 Fixed 0.1% hydrazine 242.6 Fixed 0.1% hydrazine + 2% forscorin 223.1 Fixed 100mM glycine 136.4 Fixed 100mM glycine + 2% Forscoline 391.5 Fixed 0.1% ethanolamine 232.2 Fixed 0.1% ethanolamine + 2% forscorin 75.9 Fixed 1 unit / μLCES1 857.3 Fixed 1 unit / μL CES 1 + 2% Forscorin 1458.0 Fixed 1 unit / μLCES2 316.34 Fixed 1 unit / μL CES2 + 2% Forscorin 0

[0342] Example 6: Recovery of high-quality RNA from cells fixed with compound (2a) without the use of a destabilizing agent

[0343] This example illustrates the use of compound (2a) to fix Jurkats cells, then storing them in storage buffer for up to 3 days, lysing them without the use of a destabilizing agent to extract RNA, and performing 3'-sequencing to determine the quality of the extracted RNA.

[0344] Materials and methods

[0345] A. Preparation of the stock solution:

[0346] A stock solution of the fixative for compound (2a) was prepared as described in Example 5.

[0347] Prepare PBS storage buffer solutions of sucrose, SSC, and BSA at the following concentrations. CellCover storage buffer was obtained from Anacyte Laboratories. Prepare a 50% sucrose storage buffer by dissolving sucrose in PBS to provide a final concentration of 50% w / v. Purchase a 20x SSC storage buffer from Sigma-Aldrich (Sigma catalog number S6639) and dilute it 3x in PBS before use. Prepare a 0.04% BSA storage buffer by diluting 10% BSA in PBS (Medtronic Biosciences catalog number 130-091-376) to a final concentration of 0.04%.

[0348] B. Jurkats' fixation:

[0349] The fixative was prepared by diluting the stock solution of compound (2a) in PBS to provide a concentration of 10 mM compound (2a). The prepared compound (2a) fixative was used within 10 minutes of preparation to prevent reactive imidazole-carboxylate hydrolysis. Fresh Jurkats cells were fixed in 100 μL of 10 mM compound (2a) fixative for 1 hour, followed by quenching with 10% fetal bovine serum (FBS) in PBS. The fixed cells were centrifuged at 300 g for 5 minutes to remove the supernatant, and then resuspended in 100 μL of storage buffer selected from CellCover, 50% sucrose, 3x SSC, or 0.04% BSA in PBS, and stored at 4°C for 3 days.

[0350] C. Single-cell 3′ gene expression analysis

[0351] The fixed cells in the storage buffer were centrifuged at 300g for 5 minutes to remove the supernatant, and then resuspended in 100μL PBS. The resuspended cells were then processed and sequenced according to the 10x Genomics Single Cell Gene Expression Protocol (10x Genomics, Pleasanton, California, USA).

[0352] Results: As shown in Table 5, Jurkats cells fixed with compound (2a), stored for 3 days in 3x SSC or 50% sucrose storage buffer at 4°C, and then used to prepare gene expression samples using a 10x genomics single-cell gene expression protocol, provided high-quality gene expression data comparable to those obtained from fresh cells. Notably, the Pearson correlation coefficients (R²) for fresh cells on day 0 and compound (2a)-fixed cells on day 3 in SSC or sucrose storage buffer were 0.918 and 0.897, respectively. Equally surprising was that the expression protocol did not include a destabilizing agent, but only a lysing agent. The ability to obtain high-quality gene expression data from fixed cells without the use of a destabilizing agent suggests that treatment with the fixation reagent of compound (2a) preferentially fixes cellular proteins, leading to cell degradation, while a significant amount of mRNA remains destabilized but still retained.

[0353] Table 5

[0354]

[0355] Example 7: Recovery of high-quality DNA from cells fixed with compound (2a) and defixed with CES and hydrazine

[0356] This example illustrates the use of compound (2a) to immobilize PBMCs, which are then stored in a storage buffer solution for up to 3 days, decontaminated with a mixture of CES and hydrazine, and then subjected to 3'-sequencing to determine the quality of the extracted RNA.

[0357] Materials and methods

[0358] A. Preparation of the stock solution:

[0359] The fixative for compound (2a) was prepared as described in Example 1. A 200 mM stock solution of compound (2a) was prepared in anhydrous DMSO.

[0360] Obtain a stock solution of carboxyl esterase (“CES”) at a concentration of 20 ng / L from Creative Biomart (catalog number CES1-29351TH).

[0361] A stock solution of hydrazine, a small molecule destabilizing agent, was prepared by diluting it in 50 mM Tris buffer at pH 8.3 to prepare a 10% w / v solution.

[0362] B. Storage buffer

[0363] A storage buffer for 0.04% BSA and 5% superenzyme in PBS was prepared by dissolving RNase-free BSA (catalog number B6917; Sigma-Aldrich) in PBS to a final concentration of 0.04%. The superenzyme (catalog number AM2694; Thermo Fisher Scientific) was then added to the 5% concentration.

[0364] C. PBMC Fixation

[0365] The fixative was prepared by diluting the stock solution of compound (2a) in PBS to provide a concentration of 10 mM or 20 mM compound (2a). The prepared compound (2a) fixative was used within 10 minutes of preparation to prevent reactive imidazole-carboxylic acid hydrolysis. Fresh PBMCs were fixed in 100 μL of 10 mM or 20 mM compound (2a) fixative for 1 hour, and then quenched in PBS with 3% RNase BSA. The fixed cells were centrifuged at 300 g for 5 minutes to remove the supernatant, and then resuspended for three days in 100 μL of PBS stock buffer containing 0.04% BSA and % superase at 4°C.

[0366] D. Partition and single-cell 3′ gene expression analysis

[0367] At day 0 and day 3, cells in fresh and fixed samples were collected, counted, and suspended in a standard master mixture of the single-cell 3′V3 protocol used with the Chromium system (10x Genomics, Pleasanton, CA, USA). CES was added to the same master mixture to a final concentration of 0.01 ng / μL. Simultaneously, hydrazine was added to barcode gel beads (10x Genomics, Pleasanton, CA, USA) to a final w / v concentration of 0.1%. The master mixture and gel beads were loaded into the Chromium system (10x Genomics, Pleasanton, CA, USA) to partition the samples together with the barcode gel beads into discrete droplets called GEMs (“gel beads in emulsion”). Once generated, the GEMs were collected, and a heat incubation step was performed. The heating step promotes the release of cell contents and RNA, the capture of RNA by the barcode oligonucleotides, and a reverse transcription (RT) reaction leading to cDNA synthesis, which binds the barcode to a 3′ synthon.

[0368] Automated meta-analysis of cell clusters identified using differentially expressed marker genes identifies and locates PBMC cell types in a sample. An automated script identifies PBMC cell type compositions by classifying cells based on combinations of differentially expressed known marker genes for each cell type with unclassified cells, quantifying the number and fraction of known cell types to be detected in the PBMC sample. Unclassified cells are placed into an undetermined category.

[0369] Results: As shown in Table 6, PBMCs fixed with 10 mM or 20 mM compound (2a), stored at 4°C for up to 3 days, defixed using CES and hydrazine, and then prepared and analyzed using a 10-fold genomics single-cell gene expression protocol, provided high-quality gene expression data comparable to those obtained from fresh cells. As shown in Table 6, after 3 days of fixation, each cell retained approximately 60% of the UMI count and over 90% of the median genes. Furthermore, the Pearson correlation coefficient (R²) between fresh cells on day 0 and compound (2a)-fixed cells on day 3 was [not specified in the original text].

[0370] Table 6

[0371]

[0372] In addition, such as Figure 7As described, cell counting was performed to determine the proportions of different PBMC cell types found in fresh PBMCs compared to PBMCs fixed with compound (2a), which were then stored and desoldered with CES and hydrazine. It was observed that the proportions of B cells, T cells, monocytes, and undetermined cell populations found in the fresh cell sample were similar to those found in the fixed cell sample stored for 3 days and then desoldered. These comparative PBMC cell counting results indicate that cell fixation-storage and desoldering using the compounds and methods of this disclosure can be used for the relative analysis of cell populations in fixed and stored samples in droplet-based assays.

[0373] The ability to obtain high-quality gene expression data from PBMCs fixed with compound (2a) and those fixed with CES and hydrazine demonstrates that the use of compound (2a) and the above-described defixation conditions can efficiently preserve and retrieve RNA information in PBMCs.

[0374] Despite the foregoing description or appended claims, the disclosure set forth herein is also defined by the following numbered clauses, which may be advantageous alone or in combination with one or more other clauses or implementations. Each of these individually numbered clauses may be used or combined with any of the preceding or following clauses. Therefore, these clauses are intended to support all such combinations, and are not limited to the specific combinations expressly provided below:

[0375] A method for preparing a biological sample includes contacting the sample with a first immobilization reagent composition containing a compound of formula (I).

[0376]

[0377] in:

[0378] The “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12.

[0379] 2. According to the method of Clause 1, where m = 1.

[0380] 3. The method according to any one of clauses 1-2, wherein the “connecting agent” comprises a straight-chain alkane moiety of 2-24 carbons.

[0381] 4. The method according to any one of clauses 1-3, wherein the compound of formula (I) is the compound of formula (II).

[0382]

[0383] Where n is between 1 and 13.

[0384] According to the method in Clause 4, the compound of formula (II) is selected from compounds (2a)-(2k).

[0385]

[0386]

[0387] 6. The method according to any one of Clauses 1-2, wherein the “connector” comprises an ethylene glycol portion.

[0388] 7. The method according to any one of clauses 1-2, wherein the compound of formula (I) is the compound of formula (III).

[0389]

[0390] Where n is between 1 and 12.

[0391] 8. According to the method of Clause 7, wherein the compound of formula (III) is selected from compounds (3a)-(3f)

[0392]

[0393]

[0394] 9. The method according to any one of clauses 1-2, wherein the “linker” further comprises disulfide bonds.

[0395] 10. According to the method of clauses 1-2, wherein the compound of formula (I) is the compound of formula (IV).

[0396]

[0397] Where m is from 1 to 13, and n is from 1 to 13.

[0398] 11. According to the method of Clause 10, the compound of formula (IV) is selected from compounds (4a)-(4d).

[0399]

[0400] 12. The method according to any one of clauses 1-2, wherein the “connecting agent” comprises a branched alkane portion.

[0401] 13. According to the method of Clause 12, the compound of formula (I) is compound (V):

[0402]

[0403] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0404] 14. According to the method of Clause 12, the compound of formula (V) is selected from compounds (5a)-(5c).

[0405]

[0406] 15. According to the method of Clause 12, the compound of formula (I) is compound (6):

[0407]

[0408] 16. The method according to any one of clauses 1-15, wherein the first fixative composition comprises:

[0409] (a) The concentration of the compound of formula (I) is 50 mM or less, 25 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less; and / or

[0410] (b) DMSO with a concentration of 5% or less, 2.5% or less, or 1.5% or less.

[0411] 17. The method according to any one of clauses 1-16, wherein the first fixative composition is contacted at room temperature for 3 hours or less, 60 minutes or less, 30 minutes or less, 15 minutes or less, or 5 minutes or less.

[0412] 18. The method according to any one of clauses 1-17, wherein the method further comprises contacting the sample with a second fixation reagent composition.

[0413] 19. The method according to Clause 18, wherein the second fixative composition comprises paraformaldehyde; optionally, wherein the concentration of paraformaldehyde is 1% or less. 20.

[0414] 20. The method according to Clause 18, wherein:

[0415] (a) The first immobilization reagent composition comprises a compound of formula (II).

[0416]

[0417] Where n is from 1 to 13; and

[0418] (b) The second immobilization agent composition comprises a compound of formula (III):

[0419]

[0420] Where n is between 1 and 12.

[0421] 21. The method according to Clause 18, wherein:

[0422] (a) The first immobilization agent composition comprises a compound of formula (III).

[0423]

[0424] Where n is from 1 to 12; and

[0425] (b) The second immobilization agent composition comprises a compound of formula (II):

[0426]

[0427] Where n is between 1 and 13.

[0428] 22. The method according to Clause 18, wherein:

[0429] (a) The first immobilization reagent composition comprises a compound of formula (II).

[0430]

[0431] Where n is from 1 to 13; and

[0432] (b) The second immobilization agent composition comprises a compound of formula (V):

[0433]

[0434] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0435] 23. The method according to any one of clauses 1-22, wherein the method further comprises contacting the sample with a pyrolysis agent and / or a desolvation agent.

[0436] 24. The method according to Clause 23, wherein the deconsolidating agent comprises:

[0437] (a) A compound capable of cleaving carbamate bonds; optionally, said compound is capable of cleaving carbamate bonds selected from DETA, EDA, hydrazine monohydrate, carboxylesterase or combinations thereof;

[0438] (b) A compound capable of cleaving disulfide bonds; optionally, wherein the compound capable of cleaving disulfide bonds is DTT; and / or

[0439] (c) A compound capable of reversing paraformaldehyde fixation; optionally, wherein the compound capable of reversing paraformaldehyde fixation is selected from any one of compounds (7a)-(7o).

[0440]

[0441]

[0442] 25. A composition comprising a fixed biological sample, wherein the sample comprises a cross-linked biomolecule of formula (Ia).

[0443]

[0444] in,

[0445] X 1 and X 2 It is the amine-containing portion of the same or different biomolecules in the sample; "linker" contains an ethylene glycol portion and / or a straight-chain or branched alkane portion of 2-24 carbons; m is 1 to 12.

[0446] 26. The composition according to Clause 25, wherein the “connector” comprises a straight-chain alkane moiety of 2 to 24 carbons.

[0447] 27. The composition according to Clause 25, wherein the sample comprises a cross-linked biomolecule of formula (IIa).

[0448]

[0449]

[0450] Where n is between 1 and 13.

[0451] 28. The composition according to Clause 25, wherein the “connector” comprises an ethylene glycol portion.

[0452] 29. The composition according to Clause 25, wherein the sample comprises a cross-linked biomolecule of formula (IIIa).

[0453]

[0454] Where n is between 1 and 12.

[0455] 30. The composition according to Clause 25, wherein the “connecting agent” further comprises disulfide bonds.

[0456] 31. The composition according to Clause 25, wherein the sample contains a cross-linked biomolecule of formula (IVa).

[0457]

[0458] Where m is from 1 to 13, and n is from 1 to 13.

[0459] 32. The composition according to clause 25, wherein the sample comprises a cross-linked biomolecule of formula (Va).

[0460]

[0461] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0462] 33. The composition according to clause 25, wherein the sample contains a cross-linked biomolecule of formula (VIa).

[0463]

[0464]

[0465] 34. The composition according to any one of clauses 25-33, wherein the fixed biological sample is derived from a tissue sample, a biopsy sample, or a blood sample.

[0466] 35. The composition according to clauses 25 to 34, wherein the fixed biological sample is a single cell.

[0467] 36. The compositions according to clauses 25 to 35, wherein the biological sample has been fixed with a fixation reagent composition containing a compound of formula (I).

[0468]

[0469] in:

[0470] The “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12.

[0471] 37. The composition according to Clause 36, wherein said composition comprises a compound selected from:

[0472] (a) Compound of formula (II)

[0473]

[0474] Where n is 1 to 13; optionally, the compound of formula (II) is selected from any one of compounds (2a) to (2k);

[0475] (b) Compound of formula (III)

[0476]

[0477] Where n is 1 to 12; optionally, the compound of formula (III) is selected from any one of compounds (3a) to (3f);

[0478] (c) Compound (IV)

[0479]

[0480] Where m is 1 to 13, n is 1 to 13; optionally, the compound of formula (IV) is selected from any one of compounds (4a) to (4d); and / or

[0481] (d) Compound (V)

[0482]

[0483] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0484] 38. A composition according to any one of clauses 25-37, wherein a fixed biological sample is provided in a partition; optionally, wherein the partition is in a discrete droplet.

[0485] 39. The composition according to Clause 38, wherein the partition further comprises a pyrolytic agent and / or a destabilizing agent.

[0486] 40. The composition according to clause 39, wherein the decoupling agent comprises a compound capable of cleaving a carbamate bond; optionally, said compound is capable of cleaving a carbamate bond selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof.

[0487] 41. The composition according to clauses 38-40, wherein the discrete partition further comprises beads.

[0488] 42. The composition according to Clause 41, wherein the deconsolidating agent is contained in the beads.

[0489] 43. The composition according to any one of clauses 38-42, wherein the partition further comprises an assay reagent; optionally, wherein the assay reagent is contained in the beads.

[0490] 44. The composition according to any one of clauses 38-43, wherein the partition may optionally further include a barcode, wherein the barcode is contained in the bead.

[0491] 45. A method for determination, comprising:

[0492] (a) Generate partitions containing immobilized biological samples and assay reagents, wherein the immobilized samples contain crosslinks of formula (Ia).

[0493]

[0494] in,

[0495] X 1 and X 2 It refers to the amine-containing portion of the same or different biomolecules in the sample;

[0496] The “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12;

[0497] as well as

[0498] (b) Detection of analytes from the reaction of the assay reagent and the dissolved biological sample.

[0499] 46. ​​The method according to Clause 45, wherein the partition further comprises a pyrolysis agent and / or a desolvation agent.

[0500] 47. The method according to clause 46, wherein the decongestant comprises a compound capable of cleaving urethane bonds; optionally, said compound capable of cleaving urethane bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof.

[0501] 48. The method according to Clause 46, wherein the partition further contains a pyrolysis agent but no solidification agent.

[0502] 49. A reagent kit comprising:

[0503] Assay reagents; and

[0504] Immobilization reagent compositions containing compound (I)

[0505]

[0506] in:

[0507] The “linker” comprises an ethylene glycol moiety and / or a straight-chain or branched alkane moiety of 2-24 carbons; m is 1 to 12.

[0508] 50. The kit according to claim 49, wherein the composition comprises a compound selected from:

[0509] (c) Compound of formula (II)

[0510]

[0511] Where n is 1 to 13; optionally, the compound of formula (II) is selected from any one of compounds (2a) to (2k);

[0512] (d) Compound of formula (III)

[0513]

[0514] Where n is 1 to 12; optionally, the compound of formula (III) is selected from any one of compounds (3a) to (3f);

[0515] (e) Formula (IV) compound

[0516]

[0517] Where m is 1 to 13, n is 1 to 13; optionally, the compound of formula (IV) is selected from any one of compounds (4a) to (4d); and / or

[0518] (f) Compound (V)

[0519]

[0520] R is selected from -H, -O(CO)-CH3 and -O(CO)-imidazole.

[0521] 51. The kit according to Clause 49, wherein the kit further comprises a cleavage agent and / or a deconsolidation agent.

[0522] 52. The kit according to Clause 49, wherein the kit further comprises a deconsolidating agent.

[0523] 53. The kit according to Clause 49, wherein the decongestant comprises a compound capable of cleaving carbamate bonds; optionally, said compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase or combinations thereof.

[0524] While the foregoing disclosure has been described in detail with reference to embodiments and descriptions for clarity and understanding, this disclosure includes the embodiments, descriptions, and implementations described herein for illustrative purposes and is intended as examples, and should not be construed as limiting the disclosure. It will be apparent to those skilled in the art that various modifications or changes can be made to the embodiments, descriptions, and implementations described herein, and that these modifications or changes are included within the spirit and scope of this disclosure and the appended claims. Furthermore, those skilled in the art will recognize many methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of this disclosure and covered by the appended claims.

[0525] Other embodiments of this disclosure are set forth in the following claims.

[0526] For all purposes, all disclosures in publications, patent applications, patents or other documents mentioned in this disclosure are expressly incorporated by reference to the extent that each such individual publication, patent, patent application or other document expressly indicates, for all purposes, that it is incorporated herein in its entirety by reference and is fully set forth herein. In the event of any conflict, this specification (including the specified terminology) shall prevail.

Claims

1. A compound of formula (I): in, The "linker" consists of a straight-chain alkane moiety of 2-24 carbons and a disulfide bond; and m is 1 to 12.

2. The compound according to claim 1, wherein, The compound of formula (I) is a compound of formula (IV): Where m is from 1 to 13, and n is from 1 to 13.

3. The compound according to claim 2, wherein, The compound of formula (IV) is compound (4a):

4. The compound according to claim 2, wherein, The compound of formula (IV) is compound (4b):

5. The compound according to claim 2, wherein, The compound of formula (IV) is compound (4c):

6. The compound according to claim 2, wherein, The compound of formula (IV) is compound (4d):

7. A composition comprising a fixed biological sample, wherein, The sample contains compounds of formula (IVa): in, X 1 and X 2 It is the amine-containing portion of the biomolecules in the sample; and m is between 1 and 13, and n is between 1 and 13.

8. The composition according to claim 7, wherein m = 1 and n = 1.

9. A method for preparing a biological sample, comprising contacting the biological sample with a first fixation reagent composition comprising a compound of formula (I) as defined in claim 1 or a compound of formula (IV) as defined in claim 2.

10. The method of claim 9, wherein the compound of formula (IV) is selected from compounds (4a)-(4d) as defined in any one of claims 3-6.

11. The method according to claim 9, wherein the compound of formula (IV) is compound (4a) as defined in claim 3.

12. The method according to any one of claims 9-11, wherein: (a) The first immobilization reagent composition comprises a compound of formula (I) or formula (IV) at a concentration of 50 mM or lower; and / or (b) The first fixative composition comprises a compound of formula (I) or a compound of formula (IV), and further comprises DMSO at a concentration of 5% or less.

13. The method according to any one of claims 9-11, wherein, Contact the first fixative composition with room temperature (RT) for 3 hours or less.

14. The method according to claim 12, wherein, Contact the first fixative composition with room temperature (RT) for 3 hours or less.

15. The method according to any one of claims 9-11 and 14, wherein: (a) The method further includes contacting the biological sample with a second immobilization reagent composition; and / or (b) The method further includes contacting the biological sample with a lysis agent and / or a desolvating agent.

16. The method of claim 12, wherein: (a) The method further includes contacting the biological sample with a second immobilization reagent composition; and / or (b) The method further includes contacting the biological sample with a lysis agent and / or a desolvating agent.

17. The method of claim 13, wherein: (a) The method further includes contacting the biological sample with a second immobilization reagent composition; and / or (b) The method further includes contacting the biological sample with a lysis agent and / or a desolvating agent.

18. The method according to claim 15, in, i) The second immobilization reagent composition comprises paraformaldehyde; and / or The decongestant includes: i) Compounds capable of breaking carbamate bonds; ii) Compounds capable of cleaving disulfide bonds; and / or iii) Compounds that can reverse paraformaldehyde fixation.

19. The method according to claim 16 or 17, in, i) The second immobilization reagent composition comprises paraformaldehyde; and / or The decongestant includes: i) Compounds capable of breaking carbamate bonds; ii) Compounds capable of cleaving disulfide bonds; and / or iii) Compounds that can reverse paraformaldehyde fixation.

20. The method according to claim 18, in, The concentration of paraformaldehyde is 1% or lower; The compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase or a combination thereof; Wherein, the compound capable of cleaving disulfide bonds is DTT; and / or The compound capable of reversing paraformaldehyde fixation is selected from any one of compounds (7a)-(7o).

21. The method according to claim 19, in, The concentration of paraformaldehyde is 1% or lower; The compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase or a combination thereof; Wherein, the compound capable of cleaving disulfide bonds is DTT; and / or The compound capable of reversing paraformaldehyde fixation is selected from any one of compounds (7a)-(7o).

22. A composition comprising a biological sample, wherein, The biological sample is fixed with a compound according to any one of claims 1 to 6, wherein the fixed biological sample is derived from a tissue sample, a biopsy sample, or a blood sample.

23. The composition according to claim 22, wherein, The fixed biological sample is a single cell.

24. The composition according to claim 22, wherein, The fixed biological sample is a single cell in the partition.

25. The composition according to claim 24, wherein, The partition is a discrete droplet.

26. The composition according to claim 25, in, The partition further includes: (a) Cracking agent; (b) Decontaminant; (c) a bead; and / or (d) A assay reagent.

27. The composition according to claim 26, wherein: (i) The decongestant includes compounds capable of breaking carbamate bonds; (ii) The debonding agent includes a compound capable of cleaving disulfide bonds; and / or The beads contain barcodes.

28. The composition according to claim 27, in, The compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof; and / or The compound capable of cleaving disulfide bonds is DTT.

29. The composition according to any one of claims 25-28, wherein the discrete droplets are water-in-oil droplets.

30. A method comprising: (a) Generate fixed biological samples; (b) Contacting the fixed biological sample with a reagent, wherein the fixed biological sample comprises a cross-linked biomolecule of formula (IVa); in, X 1 and X 2 It refers to the amine-containing portion of the same or different biomolecules in the biological sample; And m is from 1 to 13, and n is from 1 to 13; and (c) Detection of the analyte from the reaction of the reagent and the decomposed biological sample.

31. The method according to claim 30, in, The fixed biological sample is a single cell in the partition; and / or Where m = 1 and n = 1.

32. The method according to claim 30 or 31, wherein, (c) Including contact with pyrolysis agents and / or desolvation agents.

33. The method according to claim 32, wherein: (i) The decongestant comprises a compound capable of cleaving carbamate bonds; or (ii) The debonding agent includes a compound capable of cleaving disulfide bonds; or (iii) A cracking agent is present in the absence of a cracking agent.

34. The method according to claim 33, in, The compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof; and / or The compound capable of cleaving disulfide bonds is DTT.

35. A reagent kit comprising: Reagents; and A fixation reagent composition comprising a compound of formula (I) or formula (IV) as defined in any one of claims 1 to 6.

36. The kit according to claim 35, wherein, The kit also includes: (a) pyrolysis agent; and / or (b) Desolvating agent.

37. The kit of claim 36, wherein the deconsolidating agent comprises: (i) Compounds capable of breaking carbamate bonds; and / or (ii) Compounds capable of breaking disulfide bonds.

38. The kit according to claim 37, in, The compound capable of cleaving carbamate bonds is selected from DETA, EDA, hydrazine monohydrate, carboxylesterase, or combinations thereof; and / or The compound capable of cleaving disulfide bonds is DTT.

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