MEASUREMENT OF PROTEIN EXPRESSION USING REAGENTS WITH BARCODED OLIGONUCLEOTIDE SEQUENCES
First Claim
1. A method of measuring protein expression in cells, comprising:
- (a) contacting a plurality of oligonucleotide-conjugated antibodies with a plurality of cells comprising a plurality of protein targets, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of protein targets;
(b) partitioning the plurality of cells associated with the plurality of oligonucleotide-conjugated antibodies to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the oligonucleotide-conjugated antibodies;
(c) in the partition comprising the single cell, contacting a barcoding particle with the antibody specific oligonucleotides, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from a diverse set of unique barcode sequences;
(d) extending the oligonucleotide probes hybridized to the antibody specific oligonucleotides via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the poly(T) regions of the oligonucleotide probes to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier, or a complementary sequence thereof, and a barcode sequence; and
(e) obtaining sequence information of the plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of protein targets in one or more of the plurality of cells.
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Accused Products
Abstract
Some embodiments disclosed herein provide a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide. The oligonucleotide comprises a unique identifier for the protein binding reagent it is conjugated with, and the protein binding reagent is capable of specifically binding to a protein target. Further disclosed are methods and kits for quantitative analysis of a plurality of protein targets in a sample and for simultaneous quantitative analysis of protein and nucleic acid targets in a sample. Also disclosed herein are systems and methods for preparing a labeled biomolecule reagent, including a labeled biomolecule agent comprising a protein binding reagent conjugated with an oligonucleotide.
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Citations
20 Claims
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1. A method of measuring protein expression in cells, comprising:
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(a) contacting a plurality of oligonucleotide-conjugated antibodies with a plurality of cells comprising a plurality of protein targets, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of protein targets; (b) partitioning the plurality of cells associated with the plurality of oligonucleotide-conjugated antibodies to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the oligonucleotide-conjugated antibodies; (c) in the partition comprising the single cell, contacting a barcoding particle with the antibody specific oligonucleotides, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from a diverse set of unique barcode sequences; (d) extending the oligonucleotide probes hybridized to the antibody specific oligonucleotides via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the poly(T) regions of the oligonucleotide probes to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier, or a complementary sequence thereof, and a barcode sequence; and (e) obtaining sequence information of the plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of protein targets in one or more of the plurality of cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for simultaneous measurement of protein and gene expressions in cells, comprising:
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(a) contacting a plurality of oligonucleotide-conjugated antibodies with a plurality of cells comprising a plurality of protein targets and a plurality of mRNA target molecules, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of protein targets; (b) partitioning the plurality of cells associated with the oligonucleotide-conjugated antibodies to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the oligonucleotide-conjugated antibodies; (c) in the partition comprising the single cell, contacting a barcoding particle with the antibody specific oligonucleotides, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from a diverse set of unique barcode sequences; (d) extending the oligonucleotide probes hybridized to the antibody oligonucleotides and the mRNA target molecules via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the mRNA target molecules respectively, and the poly(T) regions of the oligonucleotide probes to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier or a sequence of an mRNA target molecule or a complementary sequence thereof, respectively, and a barcode sequence; and (e) obtaining sequence information of the plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of protein targets and the quantity of one or more of the plurality of mRNA target molecules in one or more of the plurality of cells. - View Dependent Claims (14, 15, 17, 18, 19, 20)
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16. A kit, comprising:
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a plurality of oligonucleotide-conjugated antibodies, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one protein target, and a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from at least 100 different unique barcode sequences.
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Specification