# US 2016/0258016 A1 · METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS AND SINGLE-CELL TRANSCRIPTOME PROFILING USING TEMPLATE SWITCHING REACTION

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- Number: US 2016/0258016 A1
- Document ID: US20160258016A1
- Status: Published application
- Kind: A1
- Application number: 14/912,556
- Filed: 2014-08-22
- Published: 2016-09-08
- Earliest claimed: 2013-08-23
- Assignees: Ludwig Institute for Cancer Research
- Applicants: Ludwig Institute for Cancer Research
- Inventors: Rickard Sandberg; Simone Picelli; Omid R. Faridani
- Web page: https://patentgrep.com/patent/US20160258016A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/09: Recombinant DNA-technology
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6853: using modified primers or templates
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12Q 2600/158: Expression markers
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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

This application discloses methods for cDN′A synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (INA) at the TSO 3′ end, using a methyl group donor, and/or a MgCb concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3′-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.

## Claims

### Claim 1

A method for preparing DNA that is complementary to an RNA molecule, comprising the steps of:
annealing a cDNA synthesis primer to said RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate;
conducting a reverse transcriptase reaction by contacting said RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein the TSO comprises a locked nucleic acid (LNA) at its 3′-end, under conditions suitable for extension of the first DNA strand that is complementary to the RNA molecule, rendering it additionally complementary to the TSO; and
optionally amplifying said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide using an oligonucleotide primer, wherein optionally said RNA is total RNA in a cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein said reverse transcription reaction is conducted in the presence of a methyl group donor and a metal salt.

### Claim 3 (depends on 2)

The method of claim 2, wherein said methyl group donor is betaine.

### Claim 4 (depends on 2)

The method of claim 2, wherein said metal salt is magnesium salt.

### Claim 5 (depends on 4)

The method of claim 4, wherein said magnesium salt has a concentration of at least 7 mM, at least 8 mM, or at least 9 mM.

### Claim 6 (depends on 1)

The method of claim 1, wherein said template switching oligonucleotide comprises at least one or two ribonucleotide residues and said LNA residue.

### Claim 7 (depends on 6)

The method of claim 6, wherein said at least one or two ribonucleotide residues are riboguanine.

### Claim 8 (depends on 6)

The method of claim 6, wherein said locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine, and locked 5-methylcytosine.

### Claim 9 (depends on 8)

The method of claim 8, wherein said locked nucleic acid residue is locked guanine.

### Claim 10 (depends on 1)

The method of claim 1, wherein said locked nucleic acid residue is at the 3′-most position.

### Claim 11 (depends on 1)

The method of claim 1, wherein said template switching oligonucleotide comprises at the 3′-end three nucleotide residues characterized by formula rGrG+N, wherein +N represents a locked nucleotide residue.

### Claim 12 (depends on 11)

The method of claim 11, wherein said template switching oligonucleotide comprises rGrG+G.

### Claim 13 (depends on 2)

The method of claim 2, wherein said methyl group donor is betaine, and said metal salt is MgCl2 at a concentration of at least 9 mM.

### Claim 14 (depends on 1)

The method of claim 1, further comprising amplifying said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide using an oligonucleotide primer.

### Claim 15 (depends on 1)

The method of claim 1, wherein said template switching oligonucleotide is selected from the oligonucleotides in Table S2.

### Claim 16 (depends on 1)

The method of claim 1, wherein the cDNA is synthesized on beads comprising an anchored oligo-dT primer.

### Claim 17 (depends on 16)

The method of claim 16, wherein said oligo-dT primer comprises a sequence of 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, wherein “N” is any nucleoside base, and “V” is selected from the group consisting of “A”, “C” and “G”.

### Claim 18 (depends on 14)

The method of claim 14, further comprising PCR preamplification, tagmentation, and final PCR amplification, wherein optionally the PCR preamplification is conducted without purifying the cDNA obtained from reverse transcription reaction.

### Claim 19

(canceled)

### Claim 20

(canceled)

### Claim 21 (depends on 1)

A cDNA library produced by the method of claim 1.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

A method for analyzing gene expression in a plurality of single cells, the method comprising the steps of: preparing a cDNA library according to the method of claim 1; and sequencing the cDNA library.

### Claim 24

A template switching oligonucleotide (TSO) comprising a locked nucleotide residue at its 3′-end.

### Claim 25 (depends on 24)

The TSO of claim 24, comprising three nucleotide residues at the 3′-end selected from the group consisting of +N+N+N, N+N+N, NN+N, rN+N+N, and rNrN+N, wherein N at each occurrence is independently a deoxyribonucleotide residue, rN at each occurrence is independently a ribonucleotide residue, and +N at each occurrence is independently a locked nucleotide residue.

### Claim 26 (depends on 24)

The TSO of claim 24, wherein said locked nucleotide residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine, and locked 5-methylcytosine.

### Claim 27 (depends on 25)

The TSO of claim 25, wherein said three nucleotide residues are selected from the group consisting of NN+G rNrN+G, rGrG+N, GG+N, rGrG+G, and GG+G.

### Claim 28

(canceled)

### Claim 29

(canceled)

### Claim 30

(canceled)