DIGITAL PROTEIN QUANTIFICATION
First Claim
Patent Images
1. A plurality of mixture partitions, wherein the individual mixture partitions comprise:
- i) a plurality of fixed proteins, wherein all of the fixed proteins in the individual mixture partition are from one cell; and
ii) a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins, and wherein the structurally distinct antibodies are conjugated to target-epitope specific oligonucleotides with an optionally cleavable linker, wherein the target-epitope specific oligonucleotides comprise;
a) target-epitope specific barcode sequences, wherein the target-epitope specific barcode sequences are the same for any one structurally distinct antibody and different for all other structurally distinct antibodies; and
b) optionally, unique molecular identifier sequences, wherein the optional unique molecular identifier sequences are different for every molecule of target-epitope specific oligonucleotide.
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Abstract
Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.
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Citations
31 Claims
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1. A plurality of mixture partitions, wherein the individual mixture partitions comprise:
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i) a plurality of fixed proteins, wherein all of the fixed proteins in the individual mixture partition are from one cell; and ii) a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins, and wherein the structurally distinct antibodies are conjugated to target-epitope specific oligonucleotides with an optionally cleavable linker, wherein the target-epitope specific oligonucleotides comprise; a) target-epitope specific barcode sequences, wherein the target-epitope specific barcode sequences are the same for any one structurally distinct antibody and different for all other structurally distinct antibodies; and b) optionally, unique molecular identifier sequences, wherein the optional unique molecular identifier sequences are different for every molecule of target-epitope specific oligonucleotide. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 24, 25, 26, 27, 28, 29, 30, 31)
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2. The plurality of mixture partitions, wherein the library contains at least about 10 and no more than about 10,000 structurally distinct antibodies.
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17. A plurality of mixture partitions, wherein the individual mixture partitions comprise:
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i) a plurality of fixed proteins, wherein all of the fixed proteins in the individual mixture partition are from one cell; ii) a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins; iii) a plurality of double-stranded target-epitope specific oligonucleotides, wherein the individual double-stranded target-epitope specific oligonucleotides are either covalently linked to a corresponding individual structurally distinct antibody, cleaved from the corresponding individual structurally distinct antibody, or comprise a reverse complement of an oligonucleotide covalently linked to or cleaved from the corresponding individual structurally distinct antibody, and wherein the target specific oligonucleotides comprise; a) target-epitope specific barcode sequences, wherein the target-epitope specific barcode sequences are the same for any one structurally distinct antibody and different for all other structurally distinct antibodies; b) optional unique molecular identifier sequences, wherein the unique molecular identifier sequences are different for every target-epitope specific oligonucleotide; c) a partition-specific barcode sequence that is identical among all partition-specific oligonucleotides of any one mixture partition and different from all partition-specific barcode sequences in other mixture partitions of the plurality of mixture partitions; d) a first 5′
region comprising a first sequencing primer binding region; ande) a second 5′
region comprising a second sequencing primer binding region, wherein the first and second primer binding regions are on opposite strands of the double stranded target-epitope specific oligonucleotide, are structurally different from each other, and flank the target-epitope specific barcode sequence, partition-specific barcode sequence, and optional universal molecular identifier sequence.
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18-23. -23. (canceled)
Specification