CN-122003510-A - Cell preparation compositions, systems and methods for single cell transcriptomics

Abstract

Disclosed herein are aspects of a composition generally comprising functionalized magnetic particles having an outer surface comprising a ligand thereon, wherein the ligand binds to permeabilized cells comprising a nucleic acid to produce a nucleic acid library. Also disclosed are methods of tagging at least one intracellular nucleic acid, methods of generating a nucleic acid library, systems configured for processing and practicing the disclosed compositions, and kits comprising one or more compositions for use with the disclosed method aspects.

Inventors

• B. Alves
• M. K. Hamilton
• K. Mike Kinley
• LEICK PATRICK
• A. Smirnov
• S franz

Assignees

• 生命技术公司

Dates

Publication Date

20260508

Application Date

20240816

Priority Date

20230818

Claims (20)

1. 1. A method for tagging nucleic acids within a cell, the method comprising: (a) Contacting permeabilized cells comprising at least one cellular nucleic acid with functionalized magnetic particles comprising at least one ligand, to produce a mixture comprising said permeabilized cells associated with said at least one ligand of said functionalized magnetic particles; (b) Contacting the at least one cellular nucleic acid with a first oligonucleotide, at least one nucleotide, a first primer, and a first polymerase under conditions to produce a first tagged nucleic acid strand in the permeabilized cell, and (C) Applying a magnetic field to the mixture, thereby separating the permeabilized cells and the first tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle from other components of the mixture; Wherein step (a) and step (b) can occur in either order.
2. 2. The method of claim 1, wherein the ligand comprises a carbohydrate or lectin.
3. 3. The method of claim 2, wherein the carbohydrate is selected from the group consisting of dextran, dextran-hydrogel, other dextran derivatives, chitin, chitosan, fucoidan, xylan, arabinoxylan, mannan, fucoidan, galactomannans, or derivatives thereof, or the lectin is selected from the group consisting of abrin, aggrecan, asialoglycoprotein receptor, calnexin, calreticulin, CD22, CD33, CD94, collectin (mannan binding lectin), concanavalin A, galectin, garcinia seed II lectin, leguminous plant lectin, mannose receptor, myelin-associated glycoprotein, N-acetylglucosamine receptor, phytolectin, lectin, pokeweed mitogen, ricin, selectin, sialoadhesin, soybean lectin, fagin-I, pluripotent proteoglycan, and mistletoe lectin.
4. 4. The method of claim 3, wherein the lectin is concanavalin a.
5. 5. The method of any one of claims 1 to 4, further comprising contacting the functionalized magnetic particles comprising at least one ligand with an activation buffer to increase the affinity of the permeabilized cells for the functionalized magnetic particles.
6. 6. The method of any one of claims 1 to 5, wherein the permeabilized cell is prepared by contacting the cell with a permeabilizing agent to form the permeabilized cell.
7. 7. The method of any one of claims 1 to 6, wherein the permeabilized cells are contacted with an immobilization agent or immobilized prior to permeabilizing and/or prior to contacting the permeabilized cells with the functionalized magnetic particles comprising the at least one ligand.
8. 8. The method of claim 7, further comprising providing a quenching solution to the fixative.
9. 9. The method of any one of claims 1 to 8, wherein at least one oligonucleotide is at least one nucleotide tethered to an oligonucleotide.
10. 10. The method of claim 9, wherein the oligonucleotide tethered nucleotide comprises a dideoxyadenosine triphosphate, a dideoxyguanosine triphosphate, a dideoxythymidine triphosphate, a dideoxyuridine triphosphate, a dideoxycytidine triphosphate, or any combination thereof.
11. 11. The method of claim 9 or claim 10, wherein the oligonucleotide tethered nucleotide comprises formula 1 or a salt thereof: The method comprises the steps of (1), Wherein NB is a nucleobase; Each of X and Q is independently selected from H, OH, N 3 , halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido; Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonylamino, ether, ketone, carbonyl, anhydride, ester, imide, urea, carbamate, and combinations thereof, and CXN is selected from the group consisting of alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imide, diazonium, carbamate, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and heterocyclic groups containing one to four N atoms, O atoms, S atoms, or combinations thereof.
12. 12. The method of any one of claims 1 to 11, the method further comprising: Contacting the first tagged nucleic acid strand with a second primer, wherein the second primer is partially complementary to the oligonucleotide tethered nucleotide after the first tagged nucleic acid strand is generated, to form an annealed second primer; contacting the first tagged nucleic acid strand and the annealed second primer with a second polymerase and at least one nucleotide that is not tethered to an oligonucleotide using the second primer as a template to extend the tethered oligonucleotide nucleotide sequence to form a second tagged nucleic acid strand, wherein the second tagged nucleic acid strand is present in a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle, and Placing the mixture comprising the permeabilized cells and the second tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle in a magnetic field, and separating the permeabilized cells and the second tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle from the other components of the mixture.
13. 13. The method of any one of claims 1 to 11, the method further comprising: Contacting the first tagged nucleic acid strand with a double stranded oligonucleotide, wherein after the first tagged nucleic acid strand is produced, one of the nucleotides is partially complementary to the oligonucleotide tethered nucleotide to form an annealed second primer; Ligating a non-complementary strand of said double stranded oligonucleotide to said oligonucleotide tethered nucleotide thereby producing a second tagged nucleic acid strand, wherein said second tagged nucleic acid strand is present in a mixture comprising said permeabilized cells associated with said ligand of said functionalized magnetic particle, and Placing the mixture comprising the permeabilized cells and the second tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle in a magnetic field, and separating the permeabilized cells and the second tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle from the other components of the mixture.
14. 14. The method of any one of claims 1 to 13, wherein the first primer, the second primer, and/or tethered oligonucleotide comprise a random sequence, a target-specific sequence, or both.
15. 15. The method of any one of claims 1 to 14, wherein the first primer, the second primer, or the tethered oligonucleotide comprises a universal handle, a universal sequence, a unique molecular identifier, an adapter sequence, a promoter sequence, a barcode sequence, an index sequence, or any combination thereof.
16. 16. A method for preparing a nucleic acid library, the method comprising: Preparing a sample comprising a plurality of cells, wherein at least one cell of the plurality of cells comprises a cellular nucleic acid, and wherein the at least one cell is fixed and permeabilized; Barcoding the cellular nucleic acid of the at least one immobilized and permeabilized cell in the mixture; contacting the sample with functionalized magnetic particles comprising at least one ligand, producing a mixture, wherein the at least one immobilized and permeabilized cell binds to the at least one ligand; applying a magnetic field to the sample, thereby separating the mixture from other components of the sample; Lysing the plurality of cells in the separated mixture, and A nucleic acid library is prepared from lysed cells by providing at least one amplification primer to form amplified nucleic acids.
17. 17. The method of claim 16, wherein barcoding the cellular nucleic acid comprises: Splitting the mixture into a plurality of first containers; preparing a nucleic acid complementary to the cellular nucleic acid; Annealing a first primer to form an annealed first primer at least partially complementary to at least one complementary nucleic acid, the first primer comprising a first universal handle sequence and a first barcode that is common to the containers but different from the first barcodes present in the first primer in the other containers, and Contacting the at least one complementary nucleic acid with a polymerase, at least one nucleotide, and at least one dideoxynucleotide tethered to an oligonucleotide comprising a second universal handle sequence.
18. 18. The method of claim 17, the method further comprising: Forming at least one nucleic acid strand comprising the dideoxynucleotide tethered to the oligonucleotide at its 3' end; Pooling the functionalized magnetic particles bound to the immobilized and permeabilized cells from the plurality of first containers to provide a first pool, the immobilized and permeabilized cells comprising the at least one nucleic acid strand comprising the oligonucleotide tethered dideoxynucleotide at the 3' end thereof; Washing the first tank; Splitting the first pool into a plurality of second containers; Annealing a second primer to a tethered oligonucleotide to form an annealed second primer, wherein the second primer is at least partially complementary to the tethered oligonucleotide and allows the polymerase to extend from the 3' hydroxyl of the annealed second primer to the tethered oligonucleotide to form an extended annealed second primer; pooling the functionalized magnetic particles bound to the immobilized and permeabilized cells comprising the extended annealed second primer to provide a second pool; Washing the second tank, and Splitting the second pool into a plurality of third containers.
19. 19. The method of claim 18, the method further comprising: Forming a first extension product comprising the oligonucleotide tethered dideoxynucleotide at the 3' end; Pooling the functionalized magnetic particles bound to the immobilized and permeabilized cells comprising the formed plurality of first extension products comprising the first extension product to form a third pool; Washing the third pond; Splitting the third pool into a plurality of fourth containers; contacting a splint oligonucleotide with the tethered oligonucleotide of the first extension product, wherein the splint oligonucleotide is partially complementary to the tethered oligonucleotide of the first extension product; Contacting the first extension product with a nucleic acid polymerase and one or more nucleotides to allow the polymerase to extend from the 3' hydroxyl group of the tethered oligonucleotide across the annealed splint to produce a second extension product; pooling the functionalized magnetic particles bound to the immobilized and permeabilized cells comprising the second extension product to form a fourth pool; washing the fourth tank, and Splitting the fourth pool into a plurality of fifth containers.
20. 20. The method of claim 17, the method further comprising: Forming a first extension product comprising the oligonucleotide tethered dideoxynucleotide at the 3' end; pooling the functionalized magnetic particles bound to permeabilized cells comprising the formed plurality of first extension products, the formed plurality of first extension products comprising the first extension products; washing the functionalized magnetic particles bound to permeabilized cells comprising a plurality of first extension products, said plurality of first extension products comprising said first extension products; Splitting the functionalized magnetic particles bound to permeabilized cells comprising the first extension product into a plurality of second containers; Providing a pre-annealed oligonucleotide comprising a second barcode sequence that is at least partially complementary to the tethered oligonucleotide of the first extension product; Contacting the first extension product with the pre-annealed oligonucleotide and a ligase to form a ligation product comprising the oligonucleotide tethered dideoxynucleotide at the 3' end and comprising a second barcode; pooling the functionalized magnetic particles bound to permeabilized cells comprising the ligation product; washing the functionalized magnetic particles bound to permeabilized cells comprising said ligation product, and The functionalized magnetic particles bound to permeabilized cells comprising the ligation product are split into a plurality of third containers.

Description

Cell preparation compositions, systems and methods for single cell transcriptomics Cross Reference to Related Applications The present application claims the benefit of the prior application day of U.S. provisional patent application No. 63/533,464, filed 8/18 of 2023, which is incorporated herein by reference in its entirety. Technical Field The present specification relates to compositions, systems, methods, and kits for preparing cells, such as for single cell transcriptomics. Background Single cell transcriptomics currently requires oil drop based separation techniques or laborious centrifugation. Furthermore, the current technical requirements of centrifugation of individual cells and oil droplet separation do not allow for high throughput automated methods. For example, SPLiT-seq workflow can take 10 hours or more to complete, including laborious centrifugation. SPLiT-seq is a method for single cell or nuclear transcription set analysis using cross-linking, template switching, incorporation of barcodes via ligation, and Nextera fragmentation to prepare libraries for nucleic acid sequencing. The SPLiT-seq workflow cannot be automated due to the need for multiple centrifugal washing steps. The equipment required to eliminate centrifugation or oil drop based techniques would allow high throughput automation. Thus, there is a need to eliminate costly oil drop-based separation techniques, eliminate laborious centrifugation, and reduce the workflow time required in single cell transcriptomics. In addition, current on-bead assays do not address single cell transcriptomics. For example, CUT & RUN epigenetic assays utilize target-specific primary antibodies and pAG-MNase to isolate protein DNA complexes on native chromatin for library development. In addition, CUT & TAG investigated interactions between proteins and DNA to identify binding sites for proteins of interest on DNA, based on epigenetic library development of transposases. CUTAC is another epigenetic assay, a bead-based technique for identifying open chromatin with a Tn5 transposase. Thus, there is a need for bead-on-bead methods that allow single-cell transcriptomics, as current technology only focuses on interrogation of DNA and epigenetic markers within the genome. Disclosure of Invention The disclosed aspects of the present disclosure advantageously provide advantageous aspects of a workflow for developing a nucleic acid library for single cell transcriptomics by using functionalized magnetic particles that bind to at least one permeabilized cell via a ligand. The disclosed aspects also additionally reduce the time and expense associated with developing such nucleic acid libraries. In some aspects, provided herein are methods for tagging nucleic acids within a cell, the methods comprising: (a) Contacting permeabilized cells comprising at least one cellular nucleic acid with functionalized magnetic particles comprising at least one ligand to produce a mixture comprising permeabilized cells associated with the at least one ligand of the functionalized magnetic particles; (b) Contacting at least one cellular nucleic acid with a first oligonucleotide, at least one nucleotide, a first primer, and a first polymerase under conditions to produce a first tagged nucleic acid strand in a permeabilized cell, and (C) Applying a magnetic field to the mixture, thereby separating permeabilized cells and the first tagged nucleic acid strand associated with the ligand of the functionalized magnetic particle from other components of the mixture; Wherein step (a) and step (b) can occur in either order. In some examples, the ligand of the functionalized magnetic particle is a carbohydrate or lectin. In one example, the ligand is concanavalin a. In some aspects, the method further comprises contacting the functionalized magnetic particles comprising at least one ligand with an activation buffer to increase the affinity of the permeabilized cells for the functionalized magnetic particles. In a further aspect, permeabilized cells are prepared by contacting the cells with a permeabilizing agent to form permeabilized cells. In some examples, the permeabilized cells are contacted with or immobilized with an immobilization agent prior to permeabilizing and/or prior to contacting the permeabilized cells with the functionalized magnetic particles comprising at least one ligand. The method may further comprise providing a quenching solution to the fixative. In some aspects, at least one oligonucleotide is at least one nucleotide tethered to the oligonucleotide. The nucleotides may include dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate or any combination thereof. In certain aspects, the oligonucleotide tethered nucleotide comprises formula 1 or a salt thereof: The method comprises the steps of (1), Wherein NB is a nucleobase; Each of X and Q is independently selected from H,