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US 11,891,656 B2Granted
Methods for in situ transcriptomics and proteomics
AssigneeSingular Genomics Systems, Inc.
InventorsEli N. Glezer, Hu Cang, Zhenmin Hong
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Abstract
Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.
Claims28
A method of detecting a protein and a nucleic acid molecule in a cell in situ, said method comprising:
- i) contacting the cell with a first polynucleotide probe and binding the first polynucleotide probe to the nucleic acid molecule, wherein the first polynucleotide probe comprises a first primer binding sequence and a first barcode sequence, wherein the first barcode sequence is from a known set of barcodes associated with the nucleic acid molecule;
- ii) contacting the cell with a specific binding reagent and binding the specific binding reagent to the protein, wherein the specific binding reagent comprises an oligonucleotide comprising a second primer binding sequence and a second barcode sequence associated with the specific binding reagent, wherein the second barcode sequence is from a known set of barcodes associated with the protein;
- and
- iii) hybridizing a first sequencing primer to the first primer binding sequence, and hybridizing a second sequencing primer to the second primer binding sequence, wherein the first sequencing primer and the second sequencing primer are different sequences, and
- iv) incorporating one or more modified nucleotides into the first sequencing primer with a polymerase to create a first extension strand, and detecting the one or more incorporated nucleotides in a first optically resolvable feature, and, after generating the first extension strand, incorporating one or more modified nucleotides into the second sequencing primer with a polymerase to create a second extension strand, and detecting the one or more incorporated nucleotides in a second optically resolvable feature.
2 more elements
A method of detecting a protein and a nucleic acid molecule in a cell, said method comprising:
- i) contacting the cell with a specific binding reagent and binding the specific binding reagent to the protein, wherein the specific binding reagent comprises an oligonucleotide;
- ii) contacting the cell with a first polynucleotide probe comprising a first barcode sequence, and binding the first polynucleotide probe to the nucleic acid molecule;
- iii) contacting the cell with a second polynucleotide probe comprising a second barcode sequence, and binding the second polynucleotide probe to the oligonucleotide;
- iv) amplifying the first polynucleotide probe thereby forming a first amplification product comprising the first barcode sequence, or complement thereof, and amplifying the second polynucleotide probe thereby forming a second amplification product comprising the second barcode sequence, or complement thereof;
- v) hybridizing a first sequencing primer to the first amplification product and sequencing the first barcode sequence, or a complement thereof;
- followed by hybridizing a second sequencing primer to the second amplification product and sequencing the second barcode sequence.
3 more elements
A method of detecting a protein and a nucleic acid molecule in a cell, said method comprising:
- i) contacting the cell with a specific binding reagent and binding the specific binding reagent to the protein, wherein the specific binding reagent comprises an oligonucleotide;
- ii) contacting the cell with a first polynucleotide probe comprising a first barcode sequence, and binding the first polynucleotide probe to the nucleic acid molecule;
- iii) contacting the cell with a second polynucleotide probe comprising a second barcode sequence, and binding the second polynucleotide probe to the oligonucleotide;
- iv) amplifying the first polynucleotide probe thereby forming a first amplification product comprising the first barcode sequence, or complement thereof, and amplifying the second polynucleotide probe thereby forming a second amplification product comprising the second barcode sequence, or complement thereof;
- v) hybridizing a first sequencing primer to the second amplification product and sequencing the second barcode sequence, or a complement thereof;
- followed by hybridizing a second sequencing primer to the first amplification product and sequencing the first barcode sequence, or complement thereof.
3 more elements
Dependencies are inferred from the claim text.