IP Library › Granted Patent US 12,371,733
Granted Patent B2
US 12,371,733 · App. 19/027,542 · Granted Jul 29, 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
C12Q1/6806C12N15/1065C12Q1/6855
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Quick Facts
Patent No.
US 12,371,733
App. No.
19/027,542
Granted
Jul 29, 2025
Kind
B2
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 (34)

1. A method of preparing a nucleic acid library for single-cell transcriptome analysis, the method comprising:

(a) fixing and permeabilizing a plurality of cells;

(b) generating a plurality of tagged cDNA molecules within each of the plurality of fixed and permeabilized cells;

wherein the plurality of tagged cDNA molecules generated within each of the plurality of cells corresponds to the transcriptome of the cell;

wherein the plurality of cells are present within a plurality of aliquots during the generating, with each aliquot of the plurality of aliquots comprising more than one cell; and

wherein each of the plurality of tagged cDNA molecules comprises one or more aliquot-specific barcode sequences;

(c) lysing the plurality of cells, thereby releasing the plurality of tagged cDNA molecules;

(d) isolating the released plurality of tagged cDNA molecules;

(e) amplifying the isolated plurality of tagged cDNA molecules; and

(f) sequencing the amplified plurality of tagged cDNA molecules.

2. The method of claim 1 , wherein the plurality of cells are fixed with formaldehyde.

3. The method of claim 1 , wherein generating a plurality of tagged cDNA molecules within each of the plurality of cells in step (b) comprises performing reverse transcription (RT) on the transcriptome of the cell using RT primers comprising a poly(dT) sequence.

4. The method of claim 1 , wherein generating a plurality of tagged cDNA molecules within each of the plurality of cells in step (b) comprises performing RT on the transcriptome of the cell to generate a plurality of cDNA molecules within each cell, and ligating nucleic acid tags comprising one of the one or more aliquot-specific barcode sequences to the plurality of cDNA molecules.

5. The method of claim 4 , wherein the RT is performed using RT primers comprising a 5′ terminal sequence that is the same in all of the RT primers, and wherein the nucleic acid tags each comprise a hybridization sequence that is complementary to the 5′ terminal sequence.

6. The method of claim 1 , wherein the aliquots of the plurality of aliquots are distributed in the wells of a multi-well plate.

7. The method of claim 6 , wherein the multi-well plate is a 96-well plate.

8. The method of claim 7 , wherein the sequencing of step (f) comprises sequencing the one or more aliquot-specific barcode sequences.

9. The method of claim 8 , further comprising grouping the sequencing reads generated in step (f) according to the one or more aliquot-specific barcode sequences.

10. The method of claim 9 , further comprising using the one or more aliquot-specific barcode sequences to help determine the cell origin of each of the plurality of tagged cDNA molecules.

11. The method of claim 1 , wherein the plurality of cells are lysed using a protease.

12. The method of claim 11 , wherein the protease is proteinase K.

13. The method of claim 1 , wherein the released tagged cDNA molecules are isolated in step (d) using a binding agent.

14. The method of claim 13 , wherein the binding agent comprises streptavidin beads.

15. The method of claim 1 , wherein the sequencing of step (f) is performed using next-generation sequencing (NGS).

16. The method of claim 1 , wherein the isolated tagged cDNA molecules are present within a plurality of samples during the amplifying of step (e), wherein the amplifying is performed using a plurality of amplification primers, and wherein at least a portion of the amplification primers comprise a sample-specific index sequence.

17. The method of claim 16 , wherein the sequencing of step (f) comprises sequencing the sample-specific index sequences.

18. The method of claim 17 , further comprising grouping the sequencing reads generated in step (f) according to the sample-specific index sequences.

19. The method of claim 18 , wherein the sequencing reads are grouped according to the sample-specific index sequences and the one or more aliquot-specific barcode sequences.

20. The method of claim 19 , further comprising using the sample-specific index sequences and the one or more aliquot-specific barcode sequences to help determine the cell origin of each of the plurality of tagged cDNA molecules.

21. The method of claim 1 , further comprising mapping the sequencing reads generated in step (f) to a reference genome.

22. The method of claim 21 , further comprising using the results of the mapping to determine the identity of the RNA molecule within the transcriptome that was used to generate each of the tagged cDNA molecules in step (b).

23. The method of claim 1 , wherein the plurality of cells comprises at least 2,500, 25,000, or 50,000 cells.

24. The method of claim 3 , wherein the poly(dT) sequence is a poly(dT) 15 sequence.

25. The method of claim 1 , wherein the plurality of cells comprises mammalian cells.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 17, 2025
From: SEELIG, GEORG; ROSENBERG, ALEXANDER B.; MUSCAT, RICHARD
To: UNIVERSITY OF WASHINGTON
Reel/Frame 069912/0678 →
Continuity (13)
Continuation 18887080 · Sep 17, 2024
Continuation 18784019 · Jul 25, 2024
Continuation 18753319 · Jun 25, 2024
Continuation 18455113 · Aug 24, 2023
Continuation 18158487 · Jan 24, 2023
Continuation 17814712 · Jul 25, 2022
Continuation 17695671 · Mar 15, 2022
Continuation 17521263 · Nov 8, 2021
Continuation 17249257 · Feb 25, 2021
Continuation 17122321 · Dec 15, 2020
Continuation 14941433 · Nov 13, 2015
Provisional Application 62080055 · Nov 14, 2014
Related Publication 20250163495A1 · May 22, 2025
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