Measurement of protein expression using reagents with barcoded oligonucleotide sequences

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.

1 .- 1021 . (canceled) 
     
     
         1022 . A method for measurement of cellular component target expression in cells, comprising:
 contacting a plurality of cellular component binding reagents with a plurality of cells comprising a plurality of cellular component targets, wherein each of the plurality of cellular component binding reagents comprises a cellular component binding reagent specific oligonucleotide comprising a target region, a universal sequence, and a unique identifier for the cellular component binding reagent, and wherein the cellular component binding reagent is capable of specifically binding to at least one of the plurality of cellular component targets;   partitioning the plurality of cells associated with the plurality of cellular component binding reagents 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 cellular component binding reagents;   in the partition comprising the single cell, contacting a plurality of oligonucleotide probes with the cellular component-binding reagent specific oligonucleotides, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a target binding region capable of binding to a target region, and wherein the molecular label sequence is from a diverse set of unique molecular label sequences;   extending the oligonucleotide probes hybridized to the cellular component-binding reagent specific oligonucleotides to generate a plurality of labeled nucleic acids, wherein each labeled nucleic acid comprises a unique identifier, or a complementary sequence thereof, and a molecular label sequence; and   obtaining sequence information of the plurality of labeled nucleic acids, or products thereof, to determine the copy number of at least one cellular component target of the plurality of cellular component targets in one or more of the plurality of cells.   
     
     
         1023 . The method of  claim 1022 , wherein the plurality of cellular component targets comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. 
     
     
         1024 . The method of  claim 1022 , wherein the plurality of cells comprises T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof. 
     
     
         1025 . The method of  claim 1022 , wherein the cellular component binding reagent comprises an antibody, a tetramer, a protein scaffold, or a combination thereof. 
     
     
         1026 . The method of  claim 1022 , wherein each of the oligonucleotide probes comprises a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof. 
     
     
         1027 . The method of  claim 1022 , wherein the target region comprises a poly(A) sequence. 
     
     
         1028 . The method of  claim 1022 , wherein the plurality of oligonucleotide probes are associated with a solid support, and wherein a partition of the plurality of partitions comprises a single solid support. 
     
     
         1029 . The method of  claim 1028 , wherein the solid support is a sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or a hydrogel bead. 
     
     
         1030 . The method of  claim 1022 , wherein determining the copy number of the at least one cellular component target in one or more of the plurality of cells comprises determining the copy number of the at least one cellular component target based on the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target. 
     
     
         1031 . The method of  claim 1022 , comprising removing cellular component binding reagents that are not associated with the plurality of cells. 
     
     
         1032 . The method of  claim 1022 , comprising detaching the cellular component binding reagent specific oligonucleotide from the cellular component binding reagent. 
     
     
         1033 . The method of  claim 1022 , comprising lysing the single cell in the partition comprising the single cell. 
     
     
         1034 . The method of  claim 1022 , wherein the partition is a well or a droplet. 
     
     
         1035 . The method of  claim 1022 , wherein obtaining sequencing information of the plurality of labeled nucleic acids, or products thereof, comprises subjecting the labeled nucleic acids to one or more reactions to generate a set of nucleic acids for nucleic acid sequencing. 
     
     
         1036 . The method of  claim 1022 , wherein the unique identifier comprises a nucleotide sequence of 25-45 nucleotides in length. 
     
     
         1037 . The method of  claim 1022 , wherein the unique identifier is located between the target region and the universal primer sequence. 
     
     
         1038 . The method of  claim 1022 , wherein the cellular component binding reagent specific oligonucleotide is reversibly associated with the cellular component binding reagent. 
     
     
         1039 . The method of  claim 1022 , wherein the cellular component binding reagent specific oligonucleotide is associated with the cellular component binding reagent through a chemical group selected from the group consisting of a UV photocleavable group, a streptavidin, a biotin, an amine, or a combination thereof. 
     
     
         1040 . The method of  claim 1022 , wherein the cellular component binding reagent specific oligonucleotide comprises a phosphorothioate modification. 
     
     
         1041 . The method of  claim 1022 , wherein the cellular component binding reagent specific oligonucleotide comprises a barcode sequence, and wherein at least 10 of the plurality of cellular component binding reagents each comprises a different barcode sequence.