# US 12,180,536 B2 · Methods and kits for labeling cellular molecules

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- Number: US 12,180,536 B2
- Document ID: US12180536B2
- Status: Granted
- Kind: B2
- Application number: 18/536,766
- Filed: 2023-12-12
- Published: 2024-04-04
- Granted: 2024-12-31
- Earliest claimed: 2014-11-14
- Assignees: University of Washington
- Applicants: University of Washington
- Inventors: Georg Seelig; Richard Muscat; Alexander B. Rosenberg
- Web page: https://patentgrep.com/patent/US12180536B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6869: Methods for sequencing
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## 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

### Claim 1

A method of labeling RNA molecules with cell-specific tags, the method comprising:
(a) providing a plurality of fixed and permeabilized cells, wherein each of the plurality of fixed, permeabilized cells comprises RNA;
(b) reverse transcribing RNA molecules within the cells to generate complementary DNA (cDNA) molecules, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising:
i) a poly(T) sequence or a random nucleotide sequence, and
ii) a 5′ overhang sequence located 5′ of the poly(T) or the random nucleotide sequence,
wherein the 5′ overhang sequence is the same in all of the RT primers, and
wherein following reverse transcription of the RNA, the 5′ overhang sequence is present at the 5′ end of each of the cDNA molecules;
(c) dividing the plurality of cells comprising the cDNA molecules into a plurality of aliquots, wherein each aliquot of the plurality of aliquots comprises more than one cell;
(d) coupling nucleic acid tags to the cDNA molecules within cells of the plurality of aliquots, thereby generating tagged cDNA molecules, wherein each of the nucleic acid tags comprises:
i) a barcode sequence, and
ii) a 3′ hybridization sequence located 3′ of the barcode sequence and/or a 5′ hybridization sequence located 5′ of the barcode sequence,
wherein multiple distinct barcode sequences are present among the nucleic acid tags used in the plurality of aliquots, and
wherein the barcode sequences present in each individual aliquot of the plurality of aliquots are specific to the individual aliquot;
(e) combining the cells from the plurality of aliquots;
(f) dividing the combined cells from the plurality of aliquots into a plurality of samples, wherein each sample of the plurality of samples comprises more than one cell;
(g) lysing the cells in the plurality of samples to release the tagged cDNA molecules; and
(h) amplifying the released tagged cDNA molecules in the plurality of samples using amplification primers,
wherein at least a portion of the amplification primers used in each of the plurality of samples comprise an index sequence,
wherein multiple distinct index sequences are present among the amplification primers used in the plurality of samples, and
wherein the index sequences present in each individual sample of the plurality of samples are specific to the individual sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein steps (c) through (e) are repeated one or more times, thereby generating repeatedly tagged cDNA molecules.

### Claim 3 (depends on 2)

The method of claim 2, wherein steps (c) through (e) are repeated a number of times sufficient to generate at least as many distinct combinations of barcode sequences among the repeatedly tagged cDNA molecules as the number of cells in the plurality of cells.

### Claim 4 (depends on 2)

The method of claim 2, wherein steps (c) through (e) are repeated a number of times sufficient to provide a greater than 50%, 90%, 95%, or 99% probability that the repeatedly tagged cDNAs originating from a given individual cell comprise a unique combination of barcode sequences relative to the combinations of barcode sequences present among the repeatedly tagged cDNA molecules originating from the other cells of the plurality of cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the coupling in step (d) comprises ligating a 3′ end of a nucleic acid tag to the 5′ end of a cDNA molecule.

### Claim 6 (depends on 5)

The method of claim 5, wherein the 3′ end of the nucleic acid tag is present within the 3′ hybridization sequence, and wherein the 3′ end of the nucleic acid tag is brought into proximity of the 5′ end of the cDNA molecule by being preannealed to a linker nucleic acid strand that is complementary to the 3′ hybridization sequence of the nucleic acid tag and to the 5′ overhang sequence of the RT primer.

### Claim 7 (depends on 6)

The method of claim 6, wherein the coupling is stopped by introducing a plurality of ligation stop oligos that are complementary to all or part of the linker nucleic acid strand.

### Claim 8 (depends on 2)

The method of claim 2, wherein the coupling in step (d) during the one or more times that steps (c) to (e) are repeated comprises ligating a 3′ end of a nucleic acid tag to the 5′ end of a tagged or repeatedly tagged cDNA molecule.

### Claim 9 (depends on 8)

The method of claim 8, wherein the 3′ end of the nucleic acid tag is present within the 3′ hybridization sequence, and wherein the 3′ end of the nucleic acid tag is brought into proximity of the 5′ end of the tagged or repeatedly tagged cDNA molecule by being preannealed to a secondary linker nucleic acid strand that is complementary to the 3′ hybridization sequence of the nucleic acid tag and to the 5′ hybridization sequence of a previously coupled nucleic acid tag.

### Claim 10 (depends on 9)

The method of claim 9, wherein the coupling is stopped by introducing a plurality of secondary ligation stop oligos that are complementary to all or part of the secondary linker nucleic acid strand.

### Claim 11 (depends on 1)

The method of claim 1, wherein the RT primers and/or the nucleic acid tags are DNA molecules.

### Claim 12 (depends on 1)

The method of claim 1, further comprising, prior to step (a), fixing and/or permeabilizing the plurality of cells.

### Claim 13 (depends on 2)

The method of claim 2, wherein the nucleic acid tags that are coupled to the tagged or repeatedly tagged cDNA molecules during the last of the one or more times that steps (c) to (e) are repeated comprise one or more elements selected from the group consisting of a random nucleotide sequence to prevent counting of PCR duplicates, a capture agent, and a next-generation sequencing (NGS) adapter sequence.

### Claim 14 (depends on 13)

The method of claim 13, wherein the capture agent comprises biotin.

### Claim 15 (depends on 1)

The method of claim 1, further comprising, prior to step (h), isolating the tagged cDNA released during step (g) using a binding agent.

### Claim 16 (depends on 15)

The method of claim 15, wherein the binding agent comprises streptavidin.

### Claim 17 (depends on 1)

The method of claim 1, wherein the barcode sequences present within the nucleic acid tags each comprise at least 8 nucleotides.

### Claim 18 (depends on 1)

The method of claim 1, wherein one or more of the amplification primers further comprises one or more elements selected from the group consisting of a flow-cell binding sequence, an NGS primer binding sequence, and an NGS adapter sequence.

### Claim 19 (depends on 1)

The method of claim 1, wherein the cells are lysed in step (g) in the presence of a protease.

### Claim 20 (depends on 19)

The method of claim 19, wherein the protease is proteinase K.

### Claim 21 (depends on 1)

The method of claim 1, further comprising:
(i) size selecting the tagged cDNA molecules amplified in step (h) using solid-phase reversible immobilization (SPRI) beads.

### Claim 22 (depends on 1)

The method of claim 1, further comprising:
(j) sequencing the tagged cDNA molecules amplified in step (h).

### Claim 23 (depends on 22)

The method of claim 22, further comprising:
(k) grouping the sequencing reads obtained in (j) by barcode sequence and/or index sequence.

### Claim 24 (depends on 23)

The method of claim 23, wherein steps (c) to (e) have been repeated one or more times, thereby producing repeatedly tagged cDNA molecules each comprising multiple barcode sequences, and wherein the sequencing reads are grouped by any combination of two or more sequences selected from the multiple barcode sequences and the index sequence.

### Claim 25 (depends on 1)

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

### Claim 26 (depends on 1)

The method of claim 1, wherein the RT primers each comprise a poly(T) sequence.

### Claim 27 (depends on 26)

The method of claim 26, wherein at least a portion of the poly(T) sequences present among the RT primers used in the plurality of cells comprise 15 dTs and/or are anchored poly(T) sequences.

### Claim 28 (depends on 1)

The method of claim 1, wherein the nucleic acid tags used in the plurality of aliquots comprise 96 distinct barcode sequences.

### Claim 29 (depends on 28)

The method of claim 28, wherein the plurality of aliquots comprises 96 aliquots distributed in a 96-well plate.

### Claim 30 (depends on 29)

The method of claim 29, wherein each of the 96 distinct barcode sequences is present in only one of the 96 aliquots.