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US 2024/0287591 A1Published application
Use of decoy polynucleotides in single cell multiomics
AssigneeBecton, Dickinson and Company
InventorsHye-Won SONG, Jody MARTIN, Margaret NAKAMOTO
Life of this patent
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Abstract
Disclosed herein include systems, methods, compositions, and kits for using decoy polynucleotides in noise reduction. Decoy oligonucleotides provided herein can, in some embodiments, prevent or reduce undesirable non-specific binding of intracellular target-binding reagent specific oligonucleotides or protein target-binding reagent specific oligonucleotides to non-target cellular/endogenous nucleic acids, and therefore reduce noise in methods provided herein, such as, for example, single cell multiomics analysis.
Claims89
A method for detecting protein target expression and copy number of a nucleic acid target in cells, comprising:
- fixing a plurality of cells each comprising a plurality of protein targets, copies of a nucleic acid target, and one or more non-target nucleic acid;
- permeabilizing the plurality of cells;
- contacting the plurality of cells with a blocking reagent comprising a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acid;
- contacting a plurality of protein target-binding reagents with the plurality of cells, wherein each of the plurality of protein target-binding reagents comprises an protein target-binding reagent specific oligonucleotide comprising a unique protein target identifier for the protein target-binding reagent specific oligonucleotide, and wherein the protein target-binding reagent is capable of specifically binding to at least one of the plurality of protein targets;
- partitioning the plurality of cells associated with the protein target-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 protein target-binding reagents and the cell surface target-binding reagents;
- in the partition comprising the single cell, contacting a plurality of oligonucleotide barcodes with the copies of the nucleic acid target and the protein target-binding reagent specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the protein target-binding reagent specific oligonucleotides to generate a plurality of barcoded protein target-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique protein target identifier sequence and the first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target and the first molecular label;
- obtaining sequence information of the plurality of barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in one or more of the plurality of cells;
- and
- obtaining sequence information of the plurality of barcoded protein target-binding reagent specific oligonucleotides, or products thereof, to determine the presence of at least one protein target of the plurality of protein targets in one or more of the plurality of cells.
8 more elements
A method for measuring intracellular target expression and copy number of a nucleic acid target in cells, comprising:
- fixing a plurality of cells each comprising a plurality of intracellular targets, copies of a nucleic acid target, and one or more non-target nucleic acid;
- permeabilizing the plurality of cells;
- contacting the plurality of cells with a blocking reagent comprising a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acid;
- contacting a plurality of intracellular target-binding reagents with the plurality of cells, wherein each of the plurality of intracellular target-binding reagents comprises an intracellular target-binding reagent specific oligonucleotide comprising a unique intracellular target identifier for the intracellular target-binding reagent specific oligonucleotide, and wherein the intracellular target-binding reagent is capable of specifically binding to at least one of the plurality of intracellular targets;
- partitioning the plurality of cells associated with the intracellular target-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 intracellular target-binding reagents and the cell surface target-binding reagents;
- in the partition comprising the single cell, contacting a plurality of oligonucleotide barcodes with the copies of the nucleic acid target and the intracellular target-binding reagent specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the intracellular target-binding reagent specific oligonucleotides to generate a plurality of barcoded intracellular target-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique intracellular target identifier sequence and the first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target and the first molecular label;
- obtaining sequence information of the plurality of barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in one or more of the plurality of cells;
- and
- obtaining sequence information of the plurality of barcoded intracellular target-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of at least one intracellular target of the plurality of intracellular targets in one or more of the plurality of cells.
8 more elements
A method for measuring intracellular target expression, cell surface target expression and the number of copies of a nucleic acid target in cells, comprising:
- fixing a plurality of cells comprising a plurality of intracellular targets, a plurality of cell surface targets, copies of a nucleic acid target, and one or more non-target nucleic acid;
- permeabilizing the plurality of cells;
- contacting the plurality of cells with a blocking reagent comprising a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acid;
- contacting a plurality of intracellular target-binding reagents with the plurality of cells, wherein each of the plurality of intracellular target-binding reagents comprises an intracellular target-binding reagent specific oligonucleotide comprising a unique intracellular target identifier for the intracellular target-binding reagent specific oligonucleotide, and wherein the intracellular target-binding reagent is capable of specifically binding to at least one of the plurality of intracellular targets;
- contacting a plurality of cell surface target-binding reagents with the plurality of cells associated with the intracellular target-binding reagents, wherein each of the plurality of cell surface target-binding reagents comprises an cell surface target-binding reagent specific oligonucleotide comprising a unique cell surface target identifier for the cell surface target-binding reagent specific oligonucleotide, and wherein the cell surface target-binding reagent is capable of specifically binding to at least one of the plurality of cell surface targets;
- partitioning the plurality of cells associated with the intracellular target-binding reagents and the cell surface target-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 intracellular target-binding reagents and the cell surface target-binding reagents;
- in the partition comprising the single cell, contacting a plurality of oligonucleotide barcodes with the cell surface target-binding reagent specific oligonucleotides and the intracellular target-binding reagent specific oligonucleotides and the copies of the nucleic acid target for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the intracellular target-binding reagent specific oligonucleotides to generate a plurality of barcoded intracellular target-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique intracellular target identifier sequence and the first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the cell surface target-binding reagent specific oligonucleotides to generate a plurality of barcoded cell surface target-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique cell surface target identifier sequence and the first molecular label;
- extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target and the first molecular label;
- obtaining sequence information of the plurality of barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in one or more of the plurality of cells;
- obtaining sequence information of the plurality of barcoded cell surface target-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of at least one cell surface target of the plurality of cell surface targets in one or more of the plurality of cells;
- and
- obtaining sequence information of the plurality of barcoded intracellular target-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of at least one intracellular target of the plurality of intracellular targets in one or more of the plurality of cells.
11 more elements
A kit comprising:
- a plurality of intracellular target-binding reagents, wherein each of the plurality of intracellular target-binding reagents comprises an intracellular target-binding reagent specific oligonucleotide comprising a unique intracellular target identifier for the intracellular target-binding reagent specific oligonucleotide, and wherein the intracellular target-binding reagent is capable of specifically binding to at least one intracellular target of a cell;
- a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises a first universal sequence, a cell label, a molecular label, and a target-binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences;
- a fixing agent;
- a lysis buffer;
- and/or
- a blocking reagent, wherein the blocking reagent comprises (a) a plurality of oligonucleotides capable of hybridizing to at least a portion of the intracellular target-binding reagent specific oligonucleotides, (b) comprising a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acid, or both.
3 more elements
Dependencies are inferred from the claim text.