IP Library › Granted Patent US 12,371,734
Granted Patent B2
US 12,371,734 · App. 19/027,603 · 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 12,371,734
App. No.
19/027,603
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 (28)

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) introducing into each cell of the plurality of cells a set of single-stranded nucleic acid molecules configured to hybridize to the transcriptome of each cell, under conditions such that the introduced single-stranded nucleic acid molecules hybridize to the transcriptome within each cell;

(c) introducing into each of the cells a set of nucleic acid tags configured to be coupled to the single-stranded nucleic acid molecules hybridized to the transcriptome within each cell, under conditions such that the nucleic acid tags are coupled to the single-stranded nucleic acid molecules and thereby generate a plurality of tagged nucleic acid molecules;

(d) lysing the plurality of cells;

(e) isolating the plurality of tagged nucleic acid molecules;

(f) amplifying the isolated tagged nucleic acid molecules; and

(g) sequencing the amplified tagged nucleic acid molecules.

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

3. The method of claim 1 , wherein one or more of the single-stranded nucleic acid molecules within the set comprises a poly (dT) sequence.

4. The method of claim 1 , wherein the coupling of the nucleic acid tags to the single-stranded nucleic acid molecules comprises ligation.

5. The method of claim 4 , wherein the ligation comprises covalently linking a 5′ end of a single-stranded nucleic acid molecule from the set to a 3′ end of a nucleic acid tag from the set, wherein the 5′ end of the single-stranded nucleic acid molecule and the 3′ end of the nucleic acid tag are annealed adjacently to the same nucleic acid template.

6. The method of claim 1 , wherein the plurality of cells is present within a plurality of aliquots during step (c), and wherein the set of nucleic acid tags introduced into the cells in step (c) each comprise an aliquot-specific barcode sequence.

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

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

9. The method of claim 7 , further comprising grouping the sequencing reads generated in step (g) according to the aliquot-specific barcode sequences.

10. The method of claim 1 , wherein the plurality of cells is lysed using a protease.

11. The method of claim 10 , wherein the protease is proteinase K.

12. The method of claim 1 , wherein the released tagged nucleic acid molecules are isolated in step (e) using a binding agent.

13. The method of claim 12 , wherein the binding agent comprises streptavidin beads.

14. The method of claim 1 , wherein the isolated tagged nucleic acid molecules are present within a plurality of samples during the amplifying of step (f), 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.

15. The method of claim 14 , further comprising grouping the sequencing reads generated in step (g) according to the sample-specific index sequences.

16. The method of claim 1 , further comprising extending the single-stranded nucleic acid molecules hybridized to the transcriptome using a reverse transcriptase, thereby generating a plurality of cDNA molecules within each cell corresponding to the transcriptome of the cell.

17. The method of claim 16 , wherein the sequencing of step (g) comprises sequencing the plurality of cDNA molecules.

18. The method of claim 17 , further comprising mapping the sequenced cDNA molecules to a reference genome.

19. The method of claim 18 , 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 plurality of cDNA molecules.

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

21. The method of claim 1 , wherein the plurality of cells are 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 20250163496A1 · May 22, 2025
References Cited (87)
US 7479472B1 · Harbury · 2009 [cited by applicant]
US 9938566B2 · Shepard et al. · 2018 [cited by applicant]
US 10155981B2 · Brenner · 2018 [cited by applicant]
US 10240197B1 · Brenner · 2019 [cited by applicant]
US 10626442B2 · Nolan · 2020 [cited by applicant]
US 10697013B1 · Brenner · 2020 [cited by applicant]
US 10900065B2 · Seelig · 2021 [cited by applicant]
US 10982256B2 · Nolan · 2021 [cited by applicant]
US 11512341B1 · Nolan · 2022 [cited by applicant]
US 11634752B2 · Nolan · 2023 [cited by applicant]
US 12043864B2 · Seelig · 2024 [cited by applicant]
US 20060099592A1 · Freskgard · 2006 [cited by applicant]
US 20060099626A1 · Harbury · 2006 [cited by applicant]
US 20060177833A1 · Brenner · 2006 [cited by applicant]
US 20090209430A1 · Rasmussen · 2009 [cited by applicant]
US 20090264300A1 · Franch · 2009 [cited by applicant]
US 20150329852A1 · Nolan · 2015 [cited by applicant]
US 20160122753A1 · Mikkelsen · 2016 [cited by applicant]
US 20160138086A1 · Seelig · 2016 [cited by applicant]
US 20170321251A1 · Nolan · 2017 [cited by applicant]
US 20180320241A1 · Nolan · 2018 [cited by applicant]
US 20210002733A1 · Nolan · 2021 [cited by applicant]
US 20230049314A1 · Nolan · 2023 [cited by applicant]
WO 2010094036A1 · 2010 [cited by applicant]
WO 2012106385A2 · 2012 [cited by applicant]
WO 2013036810A1 · 2013 [cited by applicant]
WO 2014201273A1 · 2014 [cited by applicant]
WO 2016100976A2 · 2016 [cited by applicant]
WO 2019236599A2 · 2019 [cited by applicant]
Adey, et al., “In vitro, long-range sequence information for de novo genome assembly via transposase contiguity,” 24(12) Genome Res. 2041-2049 (2014). [cited by applicant]
Amini, et al., “Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing,” 46(12) Nature Genetics 1343 (2014). [cited by applicant]
Brenner, et al., “In Vitro Cloning of Complex Mixtures of DNA on Microbeads: Physical Separation of Differentially Expressed cDNAs,” 97(4):1665-70 PNAS (2000). [cited by applicant]
Burton, et al., “Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions,” 31(12) Nature Biotechnology 1119 (2013). [cited by applicant]
Burzio, et al., “Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells,” 106(23):9430-34 Proc. Natl. Acad. Sci. U.S.A. (2009). [cited by applicant]
Cao, et al., Comprehensive single cell transcriptional profiling of a multicellular organism, 357(6352) Science 661 (2017). [cited by applicant]
Croucher, et al., A simple method for directional transcriptome sequencing using Illumina technology, 37(22) Nucleic Acids Research e148 (2009). [cited by applicant]
Eberwine, et al., “The promise of single-cell sequencing,” 11(1) Nat. Methods 25 (2014). [cited by applicant]
Fan, et al., “Combinatorial labeling of single cells for gene expression cytometry,” 347(6222) Science 628 (2015). [cited by applicant]
Fu, et al., Counting individual DNA molecules by the stochastic attachment of diverse labels, 108(22) PNAS 9026 (2011). [cited by applicant]
Gertz, et al., Transposase mediated construction of RNA-seq libraries, 22 Genome Research 134-141 (2011). [cited by applicant]
Gomez-Lozano, et al., “Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa,” 14(8) Env't Microbiology 2006 (2012). [cited by applicant]
Grindberg, et al., “RNA-sequencing from single nuclei,” Proc. Natl. Acad. Sci. U.S.A., 110:19802-19807 (2013). [cited by applicant]
Gupta and Gupta, “Next Generation Sequencing and Its Applications,” In: Animal Biotechnology. Models in Discovery and Translation (Ashish S. Verma and Anchal Singh, eds., Academic Press, Oxford) (2014). [cited by applicant]
Hashimshony, et al., “CEL-seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification,” Cell Reports 2:666-673 (2012). [cited by applicant]
Hoffman, et al., “DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations,” 35(13):e91 Nucleic Acids Research (2007). [cited by applicant]
Illumina TruSeq® DNA Sample Preparation Guide (2012). [cited by applicant]
Islam, et al., “Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq,” 21(7) Genome Research 1160 (2011). [cited by applicant]
Islam, et al., Quantitative single-cell RNA-seq with unique molecular identifiers, Nature Methods (2013). [cited by applicant]
Jaitin, et al., “Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types,” 343 Science 776 (2014), with 35 pages of Supplementary Material. [cited by applicant]
Lee, et al., “Highly Multiplexed Subcellular RNA Sequencing in Situ,” 343 Science 1360-1363 (2014). [cited by applicant]
Ligasova and Koberna, “In situ reverse transcription: the magic of strength and anonymity,” 38(16) Nucleic Acids Research e167 (2010). [cited by applicant]
Liu, et al., “Combinatorial Peptide Library Methods for Immunology Research,” 31:11-30 Experimental Hematology (2003). [cited by applicant]
Mamanova, et al., “FRT-seq: Amplification-free, strand-specific, transcriptome sequencing,” 7(2) Nature Methods 130 (2010). [cited by applicant]
Matsuda, et al., “Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models,” Journal of Histochemistry & Cytochemistry, 59(1):68-7… [cited by applicant]
McDavid, et al., “Data exploration, quality control and testing in single-cell qPCR,” 29(4) Bioinformatics 461 (2012). [cited by applicant]
Miller, et al., “TU-tagging: cell type specific RNA isolation from intact complex tissues,” 6(6) Nature Methods 439 (2009). [cited by applicant]
Picelli, et al., “Full-length RNA-seq from single cells using Smart-seq2,” 9(1) Nature Protocols 171 (2014). [cited by applicant]
Plessy, et al., “Population transcriptomics with single-cell resolution: A new field made possible by microfluidics,” 35 (2) Bioessays 131 (2012). [cited by applicant]
Rice, et al., “Genotoxicity of therapeutic intervention in children with acute lymphocytic leukemia,” 64(13):4464-71 Cancer Research (2004). [cited by applicant]
Rosenberg, et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding,” 360 Science 176-82 (2018). [cited by applicant]
Saliba, et al., “Single-cell RNA-seq: advances and future challenges,” Nucleic Acids Res., 42(14):8845-8860 (2014). [cited by applicant]
Schlosser, et al. “Tracing Sequence Diversity Change of RNA-Cleaving Deoxyribozymes under Increasing Selection Pressure during in Vitro Selection,” Biochemistry 2004, 43:9695-9707 (2004). [cited by applicant]
Schmid, et al., “A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing,” 7(1) PLoS One e29685 (2012). [cited by applicant]
Scott and Smith, “Searching for Peptide Ligands with an Epitope Library,” 249:386-90 (1990). [cited by applicant]
Shimizu, et al., “Qualitative De Novo Analysis of Full Length cDNA and Quantitative Analysis of Gene Expression for Common Marmoset ( [cited by applicant]
Shrivastav, et al., “Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation,” 31(1):59-70 Carcinogenesis (2009). [cited by applicant]
Staerk, et al., “Reprogramming of human peripheral blood cells to induced pluripotent stem cells,” 7(1):20-24 Cell Stem Cell (2010). [cited by applicant]
Storch, “Diagnostic virology,” 31(3):739-51 Clin. Infect. Dis. (2000). [cited by applicant]
Swaroop & Xu, “cDNA libraries from human tissues and cell lines,” 64 Cytogenetic Cell Genetics 292 (1993). [cited by applicant]
Tang, et al., “mRNA-Seq whole-transcriptome analysis of a single cell,” 6(5) Nature Methods 377 (2009). [cited by applicant]
Tang, et al., “Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system,” 48(4):287-96 Biotechniques (2010). [cited by applicant]
Treutlein, et al., “Reconstruction lineage hierarchies of the distal lung epithelium using single-cell RNA-seq,” 509 Nature 371 (2014). [cited by applicant]
Urnov, et al., “Genome editing with engineered zinc finger nucleases,” 11(9):636-46 Nature Reviews Genetics (2010). [cited by applicant]
Villegas, et al., “Expression of a novel non-coding mitochondrial RNA in human proliferating cells,” 35(21):7336-47 Nucleic Acids Res. (2007). [cited by applicant]
Vivancos, et al., “Strand-specific deep sequencing of the transcriptome,” 20 Genome Research 989 (2010). [cited by applicant]
Vo & Jedlicka, “Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for fecal and swab samples,” Molecular Ecology Resources (2014). [cited by applicant]
Watzinger, et al., “Real-Time Quantitative PCR Assays for Detection and Monitoring of Pathogenic Human Viruses in Immunosuppressed Pediatric Patients,” 42(11):5189-98 J. Clin. Microbiology (2004). [cited by applicant]
Wu, et al., “Quantitative assessment of single-cell RNA sequencing methods,” 11(1) Nature Methods 41 (2014). [cited by applicant]
Wuest, et al., “Cell-specific expression profiling of rare cell types as exemplified by its impact on our understanding of female gametophyte development,” 16(1) Current Opinion in Plant Biology 41 (2013). [cited by applicant]
Ying, et al., “Generation of Full-Length cDNA Library from Single Human Prostate Cancer Cells,” 27(3) Biotechniques 410-14 (1999). [cited by applicant]
Yozwiak, et al., “Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua,” 84(18) J. Virology 9047 (2010). [cited by applicant]
Zhang, et al., “Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection,” 3(9) Silence (2012). [cited by applicant]
Zhong, et al., High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation, Cold Spring Harbor Protocols 940 (2011). [cited by applicant]
U.S. Appl. No. 62/094,917, filed Dec. 19, 2014. [cited by applicant]
U.S. Appl. No. 62/094,919, filed Dec. 19, 2014. [cited by applicant]
U.S. Appl. No. 62/094,924, filed Dec. 19, 2014. [cited by applicant]
U.S. Appl. No. 62/080,055, filed Nov. 14, 2014. [cited by applicant]