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US 11,519,027 B2Granted
Single-cell RNA sequencing using click-chemistry
AssigneeMassachusetts Institute of Technology
InventorsPhillip A. Sharp, Dig Mahat
Life of this patent
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Abstract
The present disclosure relates to a method of sequencing nascent RNA in a cell. In some embodiments, the nascent RNA is conjugated to DNA using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Methods of the present disclosure can be used to generate genomic libraries of a cell and measure gene expression and enhancer and/or super-enhancer activity.
Claims19
A method for analyzing nascent RNA comprising:
- labeling nascent RNA in one or more permeabilized cells or nuclei by incubating the one or more permeabilized cells or nuclei with either alkyne-NTPs or azide-NTPs, which are incorporated into nascent RNA transcripts by RNA polymerases in the one or more permeabilized cells or nuclei;
- providing bead-bound or free azide-labeled single-cell barcode-adaptors if the one or more permeabilized cells or nuclei are incubated with alkyne-NTPs, or providing bead-bound or free alkyne-labeled singe-cell barcode-adaptors if the one or more permeabilized cells or nuclei are incubated with the azide-NTPs;
- contacting the lysate of the one or more permeabilized cells or nuclei with the bead-bound or free azide-labeled single-cell barcode-adaptor or the bead-bound or free alkyne-labeled single-cell barcode-adaptor in the presence of a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction reagent, forming RNA-single-cell-barcoded-adaptor conjugates;
- and
- optionally, sequencing the nascent RNA or measuring the level of RNAs by quantitative PCR (qPCR).
2 more elements
A method for analyzing nascent RNA comprising:
- labeling nascent RNA in one or more permeabilized cells or nuclei by incubating the one or more permeabilized cells or nuclei with azide-NTPs, which are incorporated into nascent RNA transcripts by RNA polymerases in the one or more permeabilized cells or nuclei;
- providing bead-bound or free alkyne-labeled single-cell barcode-adaptors;
- contacting a lysate of the one or more permeabilized cells or nuclei with the bead-bound or free alkyne-labeled single-cell barcode-adaptors in the presence of a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction reagent, forming RNA-single-cell-barcoded-adaptor conjugates;
- and
- optionally, sequencing the nascent RNA or measuring the level of RNAs by quantitative PCR (qPCR).
2 more elements
A method for analyzing RNAs in a permeabilized cell or nucleus comprising:
- transferring an azide or an alkyne group to the gamma (γ) phosphate on the 5′ end of the RNAs;
- isolating the RNAs;
- and
- optionally sequencing the RNAs;
- or measuring the level of RNAs by quantitative PCR (qPCR).
2 more elements
Dependencies are inferred from the claim text.