Quantitative massively parallel proteomics

Patent metadata

Field	Value
Publication number	US-12203939-B2
Application number	US-202017074224-A
Country	US
Kind code	B2
Filing date	Oct 19, 2020
Priority date	Jul 22, 2016
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

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.

First claim

What is claimed is:

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 via thiol/maleimide conjugation, 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, wherein each compartment further comprises an oligonucleotide comprising a unique cellular identifier sequence that corresponds to the single cell in each compartment;
c) lysing the single cells with an agent that extracts proteins and nucleic acids from the cells, wherein the nucleic acids comprise RNA and/or DNA;
d) amplifying the unique molecular identifier sequences and antibody identifier sequences in each individual compartment, using the oligonucleotide comprising the unique cellular identifier sequence;
e) analyzing the amplified unique molecular identifier sequences and antibody identifier sequences to identify the expression profile of each single cell, wherein the unique molecular identifier sequences reduce bias in the analysis; and
f) analyzing the RNA extracted from the cells to identify a transcriptomic profile of each single cell and/or the DNA extracted from the cells to obtain genomic information 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 the cellular identifier sequence corresponds to a well in a tissue culture plate.

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

5. The method of claim 4 , wherein the amplification products from the individual compartments are pooled prior to sequencing.

6. The method of claim 5 , 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.

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

8. The method of claim 1 , wherein each individual compartment further comprises a single bead that includes the oligonucleotide including the unique cellular identifier sequence that corresponds to the single cell in each compartment.

9. The method of claim 1 , wherein the unique cellular identifier oligonucleotide is between about five and about fifty nucleotides in length.

Assignees

• Verily Life Sciences Llc

Inventors

• Peikon Ian
• Tong Gary
• Liu Daniel
• Chu Ci

Classifications

• G01N2570/00

  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 title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• G01N33/58 Primary

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title