Granted Patent B2

US 12,252,730 · App. 18/304,670 · Granted Mar 18, 2025

Inventors: Georg Seelig (Seattle, WA); Alexander B. Rosenberg (Seattle, WA); Charles Roco (Seattle, WA)

Assignee: University of Washington

C12Q1/6806C12Q2521/107C12Q2525/161C12Q2543/101C12Q2563/179C12Q2563/185C12Q2565/514

Abstract

Claims (43)

1. A method of cell-specifically labeling RNA molecules within cells, the method comprising:

(a) fixing and permeabilizing a plurality of cells, wherein the plurality of cells is fixed and permeabilized at a temperature below about 8° C.;

(b) dividing the plurality of cells into a first plurality of aliquots, wherein each of the first plurality of aliquots comprises more than one cell;

(c) generating complementary DNA (cDNA) molecules within cells of the first plurality of aliquots by reverse transcribing RNA molecules within the cells, 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) an RT primer barcode sequence; and wherein

the RT primer barcode sequences present within the RT primers are specific to each aliquot of the first plurality of aliquots;

(d) pooling the cells from the first plurality of aliquots;

(e) dividing the pooled cells from the first plurality of aliquots into a second plurality of aliquots, wherein each of the second plurality of aliquots comprises more than one cell;

(f) coupling nucleic acid tags to cDNA molecules within cells of the second plurality of aliquots, wherein each of the nucleic acid tags comprises a tag barcode sequence, and wherein the tag barcode sequences present within the nucleic acid tags are specific to each aliquot of the second plurality of aliquots;

(g) pooling the cells from the second plurality of aliquots;

(h) lysing the pooled cells from the second plurality of aliquots to release the cDNA molecules and form a lysate; and

(i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.

2. The method of claim 1 , wherein before step (h), the method comprises dividing the pooled cells from the second plurality of aliquots into a plurality of samples, and wherein the method further comprises amplifying the released cDNA molecules in the plurality of samples using one or more amplification primers, wherein at least one of the amplification primers used in each sample comprises an index sequence, and wherein the index sequences present within the amplification primers are specific to each sample.

3. The method of claim 1 , further comprising:

(j) conducting a template switch of the cDNA molecules bound to the binding agent;

(k) amplifying the cDNA molecules to form an amplified cDNA molecule solution; and

(l) introducing a solid phase reversible immobilization (SPRI) bead solution to the amplified cDNA molecule solution, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is between about 0.9:1 and about 0:7:1.

4. The method of claim 3 , wherein the template switch adds a common adapter sequence to 3′ ends of the cDNA molecules.

5. The method of claim 1 , wherein the plurality of cells is fixed and permeabilized at a temperature below about 7° C., below about 6° C., below about 5° C., at about 4° C., below about 4° C., below about 3° C., below about 2° C., or below about 1° C.

6. The method of claim 1 , wherein the protease inhibitor is phenylmethanesulfonyl fluoride (PMSF) or 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF).

7. The method of claim 3 , wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is about 0.8:1.

8. The method of claim 1 , wherein steps (e) through (g) are repeated one or more times.

9. The method of claim 8 , wherein steps (e) through (g) are repeated a number of times sufficient to generate at least as many unique combinations of RT primer barcode sequences and tag barcode sequences as the total number of cells in the plurality of cells.

10. The method of claim 8 , wherein steps (e) through (g) are repeated a number of times sufficient to provide a greater than 50%, 90%, 95%, or 99% probability that the cDNA molecules in each cell are bound to a unique combination of RT primer barcode sequences and tag barcode sequences.

11. The method of claim 1 , wherein the coupling in step (f) comprises ligating 3′ ends of the nucleic acid tags to 5′ ends of the cDNA molecules.

12. The method of claim 11 , wherein the nucleic acid tags each comprise a 3′ region located 3′ to the tag barcode sequence, and wherein the 3′ ends of the nucleic acid tags are brought into proximity of the 5′ ends of the cDNA molecules by prehybridizing the nucleic acid tags with linker nucleic acid strands that are complementary to the 3′ regions of the nucleic acid tags and to 5′ terminal sequences of the cDNA molecules.

13. The method of claim 12 , wherein the RT primers each further comprise a 5′ overhang comprising a 5′ overhang sequence, and wherein the 5′ terminal sequences of the cDNA molecules comprise 5′ overhang sequences from the RT primers.

14. The method of claim 12 , wherein steps (e) through (g) are repeated one or more times, wherein the nucleic acid tags each comprise a 5′ region located 5′ to the tag barcode sequence, and wherein the 5′ terminal sequences of the cDNA molecules comprise 5′ regions from previously coupled nucleic acid tags.

15. The method of claim 12 , wherein the coupling is stopped by introducing a plurality of ligation stop oligos that are each complementary to all or part of a linker nucleic acid strand.

16. The method of claim 1 , wherein the nucleic acid tags are DNA tags.

17. The method of claim 1 , wherein steps (e) through (g) are performed one or more times, and wherein the nucleic acid tags that are coupled to the cDNA molecules during the last of the one or more times comprise a random molecular barcode, a capture agent, a flow-cell binding site, and/or a primer-binding site.

18. The method of claim 17 , wherein the capture agent comprises biotin.

19. The method of claim 1 , wherein the RT primer barcode sequences and/or the tag barcode sequences each comprise at least 8 nucleotides.

20. The method of claim 1 , wherein the plurality of cells is selected from the group consisting of mammalian cells, yeast cells, bacterial cells, and combinations thereof.

21. The method of claim 1 , wherein a portion of the RT primers comprise a poly (T) sequence and a portion of the RT primers comprise a random nucleotide sequence.

22. The method of claim 21 , wherein during step (c) the concentrations of the RT primers comprising a poly (T) sequence and the RT primers comprising a random nucleotide sequence are each between about 2.5 and 5 μM.

23. The method of claim 1 , further comprising:

(m) sequencing cDNA molecules bound to the binding agent.

24. The method of claim 23 , further comprising:

(n) grouping sequencing reads obtained in step (m) by a feature selected from the group consisting of RT primer barcode sequence, tag barcode sequence, tag barcode sequence combination, and combinations thereof.

25. The method of claim 24 , wherein the cDNA molecules have been amplified using one or more amplification primers comprising an index sequence, and wherein the grouping in step (n) further comprises grouping by index sequence.

26. The method of claim 2 , wherein the first plurality of aliquots, the second plurality of aliquots, and/or the plurality of samples are distributed in a multi-well plate.

27. The method of claim 26 , wherein the multi-well plate is a 96-well plate.

Assignments (1)

Continuity (3)

References Cited (286)

Cited By (1)

US 12,371,735