IP Library Granted Patent US 12,467,076
Granted Patent B2
US 12,467,076 · App. 19/079,081 · Granted Nov 11, 2025

Methods and kits for labeling cellular molecules

Inventors: Georg Seelig (Seattle, WA); Richard Muscat (Seattle, WA); Alexander B. Rosenberg (Seattle, WA)
Assignee: University of Washington
C12Q1/6806C12N15/1065C12Q1/6855C12Q1/6869C40B50/06
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Quick Facts
Patent No.
US 12,467,076
App. No.
19/079,081
Granted
Nov 11, 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 (21)

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

(a) fixing a plurality of cells to immobilize or hold in place components of the 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;

(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, and ligating one or more nucleic acid tags to each of the RT primers.

5 . The method of claim 4 , wherein at least a portion of the one or more nucleic acid tags comprises a next-generation sequencing (NGS) adapter sequence.

6 . The method of claim 5 , wherein the plurality of tagged cDNA molecules is amplified in step (c) using a plurality of amplification primers, and wherein at least a portion of the amplification primers comprise a homology region that is complementary to the NGS adapter sequence.

7 . The method of claim 1 , wherein the sequencing of step (d) is performed using NGS.

8 . The method of claim 1 , wherein the sequencing of step (d) comprises sequencing the barcode sequences present within the plurality of tagged cDNA molecules.

9 . The method of claim 6 , wherein at least a portion of the amplification primers comprise an index sequence, and wherein the sequencing of step (d) comprises sequencing the index sequences present within the amplified tagged cDNA molecules.

10 . The method of claim 9 , wherein the sequencing of step (d) comprises sequencing the barcode sequences and the index sequences present within the amplified tagged cDNA molecules.

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

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

13 . The method of claim 1 , wherein the plurality of cells comprise mammalian cells.

14 . The method of claim 10 , 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 Mar 19, 2025
From: SEELIG, GEORG; ROSENBERG, ALEXANDER B.; MUSCAT, RICHARD
To: UNIVERSITY OF WASHINGTON
Reel/Frame 070553/0968 →
Continuity (13)
Continuation 18887084 · 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 20250243528A1 · Jul 31, 2025
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