Methods for identifying a location of an RNA in a biological sample
Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3′ end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3′ end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.
1. A method of identifying a location of an RNA in a biological sample, the method comprising:
(a) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain and a spatial barcode;
(b) releasing the RNA from the biological sample, wherein the RNA released from the biological sample is bound by the capture domain of the capture probe;
(c) extending a 3′ end of the capture probe using the RNA bound to the capture domain as a template, thereby generating an extended capture probe hybridized to the RNA;
(d) performing random-primed DNA synthesis, thereby replacing the RNA with a second strand DNA molecule hybridized to the extended capture probe to generate a double-stranded DNA product;
(e) ligating a double-stranded sequencing adaptor to the double-stranded DNA product and
(f) determining a nucleic acid sequence of (i) all or a part of the second strand DNA molecule or a complement thereof, and (ii) the spatial barcode or a complement thereof, and using the determined nucleic acid sequences of (i) and (ii) to identify the location of the RNA in the biological sample.
2. The method of claim 1 , further comprising, between steps (c) and (d), generating one or more nick(s) in the RNA hybridized to the extended capture probe.
3. The method of claim 1 , further comprising, after step (d), performing end repair on the second strand DNA molecule hybridized to the extended capture probe to generate a blunt-ended double-stranded DNA product.
4. The method of claim 3 , further comprising adding a single adenosine (A) nucleotide to the 3′ end of the extended capture probe in the blunt-ended double-stranded DNA product.
5. The method of claim 2 , wherein the generating of the one or more nick(s) is performed using RNAse H.
6. The method of claim 1 , wherein the random-primed DNA synthesis is performed using DNA polymerase I.
7. The method of claim 4 , wherein the end repair is performed using a Klenow fragment and/or T4 DNA polymerase; the adding a single adenosine (A) nucleotide is performed using Taq polymerase; and the ligating is performed using a kinase, wherein the kinase is a polynucleotide kinase, and a DNA ligase.
8. The method of claim 1 , wherein the RNA is an mRNA.
9. The method of claim 1 , wherein the capture domain is positioned 3′ relative to the spatial barcode in the capture probe.
10. The method of claim 1 , wherein the capture domain comprises a poly(T) sequence.
11. The method of claim 1 , wherein the capture probe further comprises a unique molecular identifier and/or a cleavage domain.
12. The method of claim 3 , wherein the capture probe further comprises a cleavage domain and the method further comprises, between the ligating step and the determining step, a step of releasing the blunt-ended double-stranded DNA product from the array via cleavage of the cleavage domain.
13. The method of claim 1 , wherein the determining in step (D comprises sequencing (i) all or a part of the sequence of the RNA or a complement thereof, or (ii) the sequence of the spatial barcode or a complement thereof.
14. The method of claim 13 , wherein the sequencing is high throughput sequencing.
15. The method of claim 13 , wherein the sequencing is sequencing by ligation or hybridization of labelled probes.
16. The method of claim 1 , wherein the method further comprises, between the ligating step and the determining step, creating a library for sequencing from the double-stranded DNA product, wherein the step of creating the library comprises amplification, wherein the amplification comprises polymerase chain reaction and the method further comprises sequencing the library.
17. The method of claim 1 , wherein the releasing in step (b), comprises permeabilizing the biological sample.
18. The method of claim 1 , wherein the array is on a slide or flowcell, and wherein the slide or flowcell comprises beads and/or wells.
19. The method of claim 1 , wherein the biological sample is a tissue sample.
20. The method of claim 19 , wherein the tissue sample is a tissue section.
21. The method of claim 20 , wherein the tissue section is a fixed tissue section.
22. The method of claim 20 , wherein the tissue section is a fresh, frozen tissue section.
23. The method of claim 1 , wherein the method further comprises one or both of staining and imaging the biological sample.
24. A kit comprising:
an array comprising a plurality of capture probes, wherein a capture probe of the plurality comprises (i) a spatial barcode and (ii) a capture domain that binds to an RNA;
DNA polymerase I;
DNA ligase; and
a double-stranded sequencing adaptor.
25. The kit of claim 24 , wherein the capture domain is positioned 3′ of the spatial barcode in the capture domain.
26. The kit of claim 24 , wherein the capture probe further comprises a unique molecular identifier and/or a cleavage domain.
27. The kit of claim 24 , wherein the array is on a slide or flowcell.
28. The kit of claim 27 , wherein the slide or flowcell comprises beads or wells.
29. The kit of claim 24 , wherein the kit further comprises one or more of Taq polymerase, a polynucleotide kinase (PNK), a reverse transcriptase, a Klenow fragment, and T4 DNA polymerase.