Granted Patent
B2
US 12,305,219 · App. 18/887,080 · 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 (36)
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 correspond to the transcriptome of the cell;
wherein the plurality of fixed and permeabilized 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 from each of the plurality of cells;
(d) isolating the tagged cDNA molecules released from each of the plurality of cells;
(e) amplifying the isolated tagged cDNA molecules using a plurality of amplification primers;
wherein at least a portion of the plurality of amplification primers comprise an index sequence; and
wherein multiple index sequences are present among the plurality of amplification primers; and
(f) sequencing the amplified 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 tagged cDNA molecules in step (b) comprises performing reverse transcription (RT) within the plurality of fixed and permeabilized cells using RT primers comprising a poly(dT) sequence.
4. The method of claim 1 , wherein generating tagged cDNA molecules in step (b) comprises performing RT within the plurality of fixed and permeabilized cells using RT primers comprising a random nucleotide sequence.
5. The method of claim 1 , wherein generating tagged cDNA molecules in step (b) comprises performing RT within the plurality of fixed and permeabilized cells using RT primers, and ligating a nucleic acid tag comprising one of the one or more aliquot-specific barcode sequences to each of the RT primers.
6. The method of claim 5 , wherein the nucleic acid tags are ligated to the RT primers prior to lysing the cells in step (c).
7. The method of claim 5 , wherein the nucleic acid tags are ligated to the RT primers subsequent to lysing the cells in step (c).
8. The method of claim 1 , wherein the plurality of cells are lysed using a protease.
9. The method of claim 8 , wherein the protease is proteinase K.
10. The method of claim 1 , wherein the released tagged cDNA molecules are isolated in step (d) using a binding agent.
11. The method of claim 10 , wherein the binding agent comprises streptavidin beads.
12. The method of claim 1 , wherein the sequencing of step (f) is performed using next-generation sequencing (NGS).
13. The method of claim 1 , wherein the sequencing of step (f) comprises sequencing the one or more aliquot-specific barcode sequences.
14. The method of claim 1 , wherein the isolated tagged cDNA molecules are present within a plurality of samples during the amplifying of step (e), and wherein the index sequences present among the amplification primers are sample-specific.
15. The method of claim 14 , wherein the sequencing of step (f) comprises sequencing the sample-specific index sequences introduced into the amplified tagged cDNA sequences by the amplification primers.
16. The method of claim 15 , wherein the sequencing of step (f) comprises sequencing the one or more aliquot-specific barcode sequences and the sample-specific index sequences.
17. The method of claim 1 , wherein the plurality of cells comprises at least 2,500, 25,000, or 50,000 cells.
18. The method of claim 3 , wherein the poly(dT) sequence is a poly(dT) 15 sequence.
19. The method of claim 4 , wherein the random nucleotide sequence is a random hexamer.
20. The method of claim 1 , further comprising mapping the sequencing reads generated in step (f) to a reference genome.
21. The method of claim 16 , further comprising grouping the sequencing reads generated in step (f) using the one or more aliquot-specific barcode sequences and/or the sample-specific index sequences.
22. The method of claim 16 , further comprising using the one or more aliquot-specific barcode sequences and/or the sample-specific index sequences to help determine the cell origin of each of the plurality of tagged cDNA molecules.
23. The method of claim 1 , wherein the plurality of cells comprise mammalian cells.
24. The method of claim 1 , wherein the plurality of aliquots are distributed into the wells of a multi-well plate.
25. The method of claim 24 , wherein the multi-well plate is a 96-well plate.
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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