Granted Patent
B2
US 12,305,220 · App. 18/887,084 · Granted May 20, 2025
Methods and kits for labeling cellular molecules
Inventors:
Georg Seelig (Seattle, WA); Richard Muscat (London, GB); Alexander B. Rosenberg (Seattle, WA)
Assignee:
University of Washington
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Abstract
Methods of uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within 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, cDNAs, DNAs, proteins, peptides, and/or antigens.
Claims (26)
1. A method of preparing a nucleic acid library for single-cell transcriptome analysis, the method comprising:
(a) fixing a plurality of cells;
(b) generating a plurality of tagged cDNA molecules from the transcriptome of each cell of the plurality of fixed cells,
wherein the plurality of tagged cDNA molecules generated from the transcriptome of each cell corresponds to the transcriptome of the cell; and
wherein each tagged cDNA molecule of the plurality of tagged cDNA molecules comprises a barcode sequence corresponding to the cell of origin of the tagged cDNA molecule;
(c) amplifying the plurality of tagged cDNA molecules generated from the transcriptome of each cell using a plurality of amplification primers,
wherein the plurality of amplification primers comprises forward and reverse amplification primers,
wherein at least a portion of the forward amplification primers or at least a portion of the reverse amplification primers comprise an index sequence, and
wherein multiple index sequences are present among the forward amplification primers or among the reverse amplification primers;
(d) sequencing the amplified tagged cDNA molecules; and
(e) mapping the sequencing reads obtained in (d) to a reference genome.
2. The method of claim 1 , wherein the plurality of cells are fixed with formaldehyde.
3. The method of claim 1 , wherein generating tagged cDNA molecules from the transcriptome of each cell comprises performing reverse transcription (RT) using RT primers comprising a poly(dT) sequence.
4. The method of claim 1 , wherein generating tagged cDNA molecules from the transcriptome of each cell comprises performing RT using RT primers each comprising a random nucleotide sequence.
5. The method of claim 1 , wherein generating tagged cDNA molecules from the transcriptome of each cell comprises performing RT using RT primers, and ligating one or more nucleic acid tags to each of the RT primers.
6. The method of claim 5 , wherein at least a portion of the one or more nucleic acid tags comprises a next-generation sequencing (NGS) adapter sequence.
7. The method of claim 6 , wherein at least a portion of the amplification primers comprise a homology region that is complementary to the NGS adapter sequence.
8. The method of claim 1 , wherein the sequencing of step (d) is performed using NGS.
9. The method of claim 1 , wherein the sequencing of step (d) comprises sequencing the barcode sequences present within the plurality of tagged cDNA molecules.
10. The method of claim 1 , wherein the sequencing of step (d) comprises sequencing the index sequences introduced into the amplified tagged cDNA molecules by the amplification primers.
11. The method of claim 1 , wherein the sequencing of step (d) comprises sequencing the barcode sequences present within each of the plurality of tagged cDNA molecules and the index sequences introduced into the amplified tagged cDNA molecules by the amplification primers.
12. The method of claim 3 , wherein the poly(dT) sequence is a poly(dT) 15 sequence.
13. The method of claim 4 , wherein the random nucleotide sequence is a random hexamer.
14. The method of claim 1 , wherein the plurality of cells comprises at least 2,500, 25,000, or 50,000 cells.
15. The method of claim 1 , wherein the plurality of cells comprise mammalian cells.
16. The method of claim 11 , further comprising grouping the sequencing reads generated in step (d) using the barcode sequences and/or the index sequences.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 17, 2024
From: SEELIG, GEORG; ROSENBERG, ALEXANDER B.; MUSCAT, RICHARD
To: UNIVERSITY OF WASHINGTON
Reel/Frame 068604/0870 →
Continuity (12)
Provisional Application
62080055
· Nov 14, 2014
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