MEASUREMENT OF PROTEIN EXPRESSION USING REAGENTS WITH BARCODED OLIGONUCLEOTIDE SEQUENCES
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 composition, comprising:
a cell associated with a first cellular component binding reagent,
wherein the cell comprises a first cellular component target,
wherein the first cellular component binding reagent is specifically bound to the first cellular component target, and
wherein the first cellular component binding reagent comprises a first cellular component binding reagent specific oligonucleotide comprising a target region, a universal sequence, and a unique identifier for the first cellular component binding reagent.
1023 .) The composition of claim 1022 , wherein the cell is a single cell situated in a partition.
1024 . The composition of claim 1023 , wherein the partition comprises a plurality of oligonucleotide probes, wherein at least 100 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.
1025 . The composition of claim 1024 , wherein at least 100 of the plurality of oligonucleotide probes comprises a cell label, a universal sequence, an amplification adaptor, a sequencing adaptor, or a combination thereof.
1026 . The composition of claim 1022 , wherein the target region comprises a poly(A) sequence.
1027 . The composition of claim 1024 , wherein the plurality of oligonucleotide probes are associated with a solid support, and wherein the partition comprises a single solid support and the single cell.
1028 . The composition of claim 1027 , 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.
1029 . The composition of claim 1027 , wherein the solid support is disruptable upon application of a chemical stimulus, and wherein the partition comprises the chemical stimulus.
1030 . The composition of claim 1023 , wherein the partition is a well or a droplet.
1031 . The composition of claim 1022 , wherein the first cellular component target 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.
1032 . The composition of claim 1022 , wherein the cell is a T cell, a B cell, a tumor cell, a myeloid cell, a blood cell, a normal cell, a fetal cell, a maternal cell, or a combination thereof.
1033 . The composition of claim 1022 , wherein the first cellular component binding reagent comprises an antibody, a tetramer, a protein scaffold, or a combination thereof.
1034 . The composition of claim 1022 , wherein the first cellular component binding reagent specific oligonucleotide is reversibly associated with the first cellular component binding reagent.
1035 . The composition of claim 1022 , wherein the first cellular component binding reagent specific oligonucleotide is associated with the first 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.
1036 . The composition of claim 1022 , wherein the first cellular component binding reagent specific oligonucleotide comprises a phosphorothioate modification.
1037 . The composition of claim 1022 , wherein the cell comprises a second cellular component target, wherein the cell is associated with a second cellular component binding reagent, wherein the second cellular component binding reagent is specifically bound to a second cellular component target, and wherein the second cellular component binding reagent is not associated with an oligonucleotide.
1038 . The composition of claim 1037 , wherein:
the first cellular component target and the second cellular component target are the same; and/or
the first cellular component binding reagent and the second cellular component binding reagent are the same.
1039 . The composition of claim 1022 , wherein the partition does not comprise a first cellular component binding reagent not associated with the cell.
1040 . The composition of claim 1022 , wherein the cell comprises a third cellular component target, wherein the cell is associated with a third cellular component binding reagent, wherein the third cellular component binding reagent is specifically bound to the third cellular component target, and wherein the third cellular component binding reagent comprises a third cellular component binding reagent specific oligonucleotide comprising a target region, a universal sequence, and a unique identifier for the third cellular component binding reagent, and wherein the first cellular component target and the third cellular component target are different.
1041 . The composition of claim 1022 , wherein:
the unique identifier comprises a nucleotide sequence of 25-45 nucleotides in length; and/or
is located between the target region and the universal sequence.