QUANTITATIVE MASSIVELY PARALLEL PROTEOMICS

• US 20180024139A1
• Filed: 07/21/2017
• Published: 01/25/2018
• Est. Priority Date: 07/22/2016
• Status: Active Grant

• Current Assignee: Verily Life Sciences LLC (Alphabet Inc.)
• Original Assignee: Verily Life Sciences LLC (Alphabet Inc.)
• Inventors: Peikon, Ian, Tong, Gary, Liu, Daniel, Chu, Ci
• Granted Patent: US 10,809,266 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 2570/00 Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Provided herein are compositions and methods for the identification of an expression profile in a single cell or population of cells. Kits for use with the disclosed methods are also provided, including antibodies, with a unique molecular identifier and antibody identifier, and primers for amplification of the antibody identifier sequence.

Claims

1. A method of identifying an expression profile in a single cell of a population of cells, comprising:

a) contacting a population of cells with a plurality of antibodies under conditions that promote specific binding of the antibodies to target antigens of the cells, wherein each antibody in the plurality is capable of binding to a unique target antigen in one or more cells, wherein each antibody is conjugated to an oligonucleotide, and wherein the oligonucleotide comprises a unique molecular identifier sequence and an antibody identifier sequence corresponding to the unique target antigen;

b) separating single cells of the population into individual compartments;

c) amplifying the unique molecular identifier sequences and antibody identifier sequences in each individual compartment; and

d) analyzing the amplified unique molecular identifier sequences and antibody identifier sequences to identify the expression profile of each single cell.

2. The method of claim 1, wherein the population of cells is fixed and permeabilized prior to contacting the population with the plurality of antibodies.

3. The method of claim 1, wherein each compartment further comprises an oligonucleotide comprising a unique cellular identifier sequence that corresponds to the single cell in each compartment.

4. The method of claim 3, wherein the unique molecular identifier sequence and antibody identifier sequence in each individual compartment are amplified using the oligonucleotide comprising the unique cellular identifier sequence that corresponds to the single cell in each individual compartment.

5. The method of claim 3, wherein the cellular identifier sequence corresponds to a well in a tissue culture plate.

6. The method of claim 1, wherein analyzing comprises sequencing the amplified unique molecular identifier sequences and antibody identifier sequences.

7. The method of claim 1, wherein the amplification products from the individual compartments are pooled prior to sequencing.

8. The method of claim 7, wherein the pooled sequencing results are deconvoluted using the unique cellular identifier sequences to identify the expression profile of a single cell associated with each unique cellular identifier sequence.

9. The method of claim 1, wherein the individual compartments are wells of a tissue culture plate or microfluidic droplets.

10. A method of identifying an expression profile of a population of cells, comprising:

a) contacting a population of cells with a plurality of antibodies under conditions that promote specific binding of the antibodies to the cells;

wherein each antibody in the plurality is capable of binding to a unique target antigen in one or more cells, wherein each antibody is conjugated to an oligonucleotide, and wherein the oligonucleotide comprises a unique molecular identifier sequence and an antibody identifier sequence corresponding to the unique target antigen;

b) amplifying the unique molecular identifier sequences and antibody identifier sequences; and

c) analyzing the amplified unique molecular identifier sequences and antibody identifier sequences to identify the expression profile of the population of cells.

11. The method of claim 10, wherein analyzing comprises sequencing the amplified unique molecular identifier sequences and antibody identifier sequences.

12. A method of identifying an expression profile of a population of cells, comprising:

a) lysing a population of cells;

b) labeling the cellular proteins in the cell lysate of step a) with an affinity label;

c) contacting the affinity labeled proteins with beads comprising an agent that binds the affinity label to generate protein-coated beads;

d) contacting the protein-coated beads with a plurality of antibodies under conditions that promote specific binding of the antibodies to the protein-coated beads;

wherein each antibody in the plurality binds to a unique target protein, wherein each antibody is conjugated to an oligonucleotide, and wherein the oligonucleotide comprises a unique molecular identifier sequence and an antibody identifier sequence corresponding to the unique target protein;

e) amplifying the unique molecular identifier sequences and antibody identifier sequences; and

f) analyzing the amplified unique molecular identifier sequences and antibody identifier sequences to identify the expression profile of the population of cells.

13. The method of claim 12, wherein single cells of the population are separated into individual compartments prior to the lysing of the cells.

14. The method of claim 12, wherein each compartment further comprises an oligonucleotide comprising a unique cellular identifier sequence that corresponds to the single cell in each compartment.

15. The method of claim 12, wherein the antibody identifier sequence in each individual compartment is amplified using the oligonucleotide comprising the unique cellular identifier sequence that corresponds to the single cell in each individual compartment.

16. The method of claim 12, wherein the cellular identifier sequence corresponds to a well in a tissue culture plate.

17. The method of claim 12, wherein analyzing comprises sequencing the amplified unique molecular identifier sequence and antibody identifier sequences.

18. The method of claim 17, wherein the amplification products from the individual compartments are pooled prior to sequencing.

19. The method of claim 18, wherein the pooled sequencing results are deconvoluted using the unique cellular identifier sequences to identify the expression profile of a single cell associated with each unique cellular identifier sequence.

20. The method of claim 12, wherein the individual compartments are wells of a tissue culture plate or microfluidic droplets.