Digital protein quantification
View Patent ↗Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.
1 . A method comprising:
incubating a plurality of permeabilized cells with a plurality of structurally distinct binding elements, each binding element having a specific binding affinity for a target epitope of a cellular protein, and wherein each binding element is conjugated to an epitope-specific oligonucleotide (ESO) comprising a barcode sequence specific to the target epitope, thereby binding the binding elements to their corresponding epitopes to form binder-epitope complexes;
washing away unbound binding elements;
isolating single cells of the plurality of permeabilized cells into a partition of a plurality of partitions;
providing to partitions of the plurality of partitions a plurality of partition-specific oligonucleotides (PSO) linked to a bead, each PSO comprising a partition-specific barcode sequence and a unique molecular identifier (UMI); and
hybridizing, in one or more partitions, a PSO with an ESO and extending the hybridized PSO with a polymerase, thereby generating a nucleic acid comprising an ESO, a partition-specific barcode sequence, and a UMI.
2 . The method of claim 1 , wherein each partition-specific oligonucleotide further comprises a primer binding sequence.
3 . The method of claim 1 , wherein each binding element is an antibody.
4 . The method of claim 1 , wherein each binding element is an aptamer.
5 . The method of claim 1 , wherein the plurality of structurally distinct binding elements comprises a library of at least 10, and no more than about 10,000, structurally distinct antibodies conjugated to the epitope-specific oligonucleotides.
6 . The method of claim 1 , further comprising discarding partitions that do not contain a single cell or contain multiple cells.
7 . The method of claim 1 , further comprising discarding partitions that do not contain a single partition-specific barcode sequence or contain multiple partition-specific barcode sequences.
8 . The method of claim 1 , wherein the partition-specific oligonucleotides are covalently linked to a bead with a cleavable linker.
9 . The method of claim 1 , further comprising lysing the cells after the isolating step.
10 . The method of claim 1 , wherein the plurality of partitions comprise a primer that binds to a primer binding sequence.
11 . The method of claim 10 , wherein the primer binding sequence is within the epitope-specific oligonucleotides.
12 . The method of claim 1 , wherein the epitope-specific oligonucleotide comprises a unique molecular identifier.
13 . The method of claim 1 , wherein each binding element is conjugated to a unique molecular identifier (UMI).
14 . The method of claim 1 , wherein each epitope-specific oligonucleotide further comprises a universal primer binding site.
15 . The method of claim 1 , further comprising cleaving the epitope-specific oligonucleotide from each binding element to yield a cleaved antibody oligonucleotide and cleaving the partition-specific oligonucleotide linked to the bead to yield a cleaved partition-specific oligonucleotide.
16 . The method of claim 15 , further comprising amplifying the cleaved antibody oligonucleotide using a universal primer and the cleaved partition-specific oligonucleotide.
17 . The method of claim 1 , wherein the plurality of partitions comprise wells, microwells or nanowells.
18 . The method of claim 1 , wherein the plurality of partitions comprise droplets.
19 . The method of claim 10 , wherein the primer binding sequence is within the partition-specific oligonucleotides.