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US 2020/0263234 A1Published application
In situ combinatorial labeling of cellular molecules
AssigneeUniversity of Washington
InventorsGeorg SEELIG, Alexander B. ROSENBERG, Charles ROCO
Life of this patent
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Abstract
Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.
Claims27
A method of uniquely labeling RNA molecules within a plurality of cells, the method comprising:
- (a) fixing and permeabilizing a first plurality of cells prior to step (b), wherein the first plurality of cells are fixed and permeabilized at below about 8° C.;
- (b) reverse transcribing the RNA molecules within the first plurality of cells to form complementary DNA (cDNA) molecules within the first plurality of cells, wherein reverse transcribing the RNA molecules comprises coupling primers to the RNA molecules, wherein the primers comprise at least one of a poly(T) sequence or a random sequence;
- (c) dividing the first plurality of cells comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot;
- (d) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tags provided to the first primary aliquot are different from the primary nucleic acid tags provided to the second primary aliquot;
- (e) coupling the cDNA molecules within each of the at least two primary aliquots with the provided primary nucleic acid tags;
- (f) combining the at least two primary aliquots;
- (g) dividing the combined primary aliquots into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot;
- (h) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tags provided to the first secondary aliquot are different from the secondary nucleic acid tags provided to the second secondary aliquot;
- (i) coupling the cDNA molecules within each of the at least two secondary aliquots with the provided secondary nucleic acid tags;
- (j) repeating steps (f), (g), (h), and (i) with subsequent aliquots, wherein the final nucleic acid tags comprise a capture agent;
- (k) combining final aliquots;
- (l) lysing the first plurality of cells to release the cDNA molecules from within the first plurality of cells to form a lysate;
- and
- (m) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.
11 more elements
5-6. (canceled)
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20-21. (canceled)
A method of labeling nucleic acids within a first cell, the method comprising:
- (a) generating complementary DNA (cDNA) molecules within a plurality of cells comprising the first cell by reverse transcribing RNAs using at least one of a:
- (i) a first reverse transcription primer comprising a 5′ overhang sequence, wherein the first reverse transcription primer is configured to reverse transcribe RNA having a poly(A) tail;
- and
- (ii) a second reverse transcription primer comprising a 5′ overhang sequence and at least one of a random hexamer, a random septamer, a random octomer, a random nonamer, and a random decamer;
- (b) dividing the plurality of cells into a number (n) of aliquots;
- (c) providing a plurality of nucleic acid tags to each of the n aliquots, each nucleic acid tag comprising:
- a first strand comprising a 3′ hybridization sequence extending from a 3′ end of a labeling sequence and a 5′ hybridization sequence extending from a 5′ end of the labeling sequence, and
- a second strand comprising an overhang sequence, the overhang sequence comprising
- (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhang sequence, and
- (ii) a second portion complementary to the 3′ hybridization sequence, wherein each labeling sequence of the plurality of nucleic acid tags provided into a given aliquot is the same, and wherein a different labeling sequence is provided into each of the n aliquots;
- (d) binding at least one of the cDNA molecules in each of the n aliquots to the nucleic acid tags;
- (e) combining the n aliquots;
- and
- (f) repeating steps (b), (c), (d), and (e) with the combined aliquot;
- (g) combining final aliquots;
- (h) lysing the plurality of cells comprising the first cell to release the cDNA molecules from within the plurality of cells comprising the first cell to form a lysate;
- and
- (i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.
15 more elements
23-44. (canceled)
A kit for labeling nucleic acids within a first cell, the kit comprising:
- a first reverse transcription primer comprising a 5′ overhang sequence, wherein the first reverse transcription primer is configured to reverse transcribe RNA having a poly(A) tail;
- a second reverse transcription primer comprising a 5′ overhang sequence and at least one of a random hexamer, a random septamer, a random octomer, a random nonamer, and a random decamer;
- a plurality of first nucleic acid tags, wherein each first nucleic acid tag comprises:
- a first strand comprising a 3′ hybridization sequence extending from a 3′ end of a first labeling sequence and a 5′ hybridization sequence extending from a 5′ end of the first labeling sequence, and
- a second strand comprising an overhang sequence, the overhang sequence comprising (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhang sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3′ hybridization sequence;
- a plurality of second nucleic acid tags, wherein each second nucleic acid tag comprises:
- a first strand comprising a 3′ hybridization sequence extending from a 3′ end of a second labeling sequence and a 5′ hybridization sequence extending from a 5′ end of the second labeling sequence, and
- a second strand comprising an overhang sequence, the overhang sequence comprising (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhang sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3′ hybridization sequence, wherein the first labeling sequence is different from the second labeling sequence;
- and
- a plurality of final nucleic acid tags, wherein each final nucleic acid tag comprises:
- a first strand comprising a 3′ hybridization sequence extending from a 3′ end of a final labeling sequence and a 5′ hybridization sequence extending from a 5′ end of the final labeling sequence,
- a second strand comprising an overhang sequence, the overhang sequence comprising (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhang sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3′ hybridization sequence, and
- a capture agent, wherein the final labeling sequence is different from the first and second labeling sequences.
10 more elements
46-47. (canceled)
A method of uniquely labeling RNA molecules within a plurality of nuclei, the method comprising:
- (a) fixing and permeabilizing a first plurality of nuclei prior to step (b), wherein the first plurality of nuclei are fixed and permeabilized at below about 8° C.;
- (b) reverse transcribing the RNA molecules within the first plurality of nuclei to form complementary DNA (cDNA) molecules within the first plurality of nuclei, wherein reverse transcribing the RNA molecules comprises coupling primers to the RNA molecules, wherein the primers comprise at least one of a poly(T) sequence or a random sequence;
- (c) dividing the first plurality of nuclei comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot;
- (d) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tags provided to the first primary aliquot are different from the primary nucleic acid tags provided to the second primary aliquot;
- (e) coupling the cDNA molecules within each of the at least two primary aliquots with the provided primary nucleic acid tags;
- (f) combining the at least two primary aliquots;
- (g) dividing the combined primary aliquots into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot;
- (h) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tags provided to the first secondary aliquot are different from the secondary nucleic acid tags provided to the second secondary aliquot;
- (i) coupling the cDNA molecules within each of the at least two secondary aliquots with the provided secondary nucleic acid tags;
- (j) repeating steps (f), (g), (h), and (i) with subsequent aliquots, wherein the final nucleic acid tags comprise a capture agent;
- (k) combining final aliquots;
- (l) lysing the first plurality of nuclei to release the cDNA molecules from within the first plurality of nuclei to form a lysate;
- and
- (m) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.
11 more elements
49-67. (canceled)
A method of labeling nucleic acids within a first nucleus, the method comprising:
- (a) generating complementary DNA (cDNA) molecules within a plurality of nuclei comprising the first nucleus by reverse transcribing RNAs using at least one of a:
- a first reverse transcription primer comprising a 5′ overhang sequence, wherein the first reverse transcription primer is configured to reverse transcribe RNA having a poly(A) tail;
- and
- (ii) a second reverse transcription primer comprising a 5′ overhang sequence and at least one of a random hexamer, a random septamer, a random octomer, a random nonamer, and a random decamer;
- (b) dividing the plurality of nuclei into a number (n) of aliquots;
- (c) providing a plurality of nucleic acid tags to each of the n aliquots, each nucleic acid tag comprising:
- a first strand comprising a 3′ hybridization sequence extending from a 3′ end of a labeling sequence and a 5′ hybridization sequence extending from a 5′ end of the labeling sequence, and
- a second strand comprising an overhang sequence, the overhang sequence comprising (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhang sequence, and (ii) a second portion complementary to the 3′ hybridization sequence,
- wherein each labeling sequence of the plurality of nucleic acid tags provided into a given aliquot is the same, and wherein a different labeling sequence is provided into each of the n aliquots;
- (d) binding at least one of the cDNA molecules in each of the n aliquots to the nucleic acid tags;
- (e) combining the n aliquots;
- and
- (f) repeating steps (b), (c), (d), and (e) with the combined aliquot;
- (g) combining final aliquots;
- (h) lysing the plurality of nuclei comprising the first nucleus to release the cDNA molecules from within the plurality of nuclei comprising the first nucleus to form a lysate;
- and
- (i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.
14 more elements
69-89. (canceled)
Dependencies are inferred from the claim text.