Chemical compositions and uses thereof
The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell that are adaptable for use with existing sequencing technologies.
1. A method comprising:
a) collecting a plurality of oligonucleotides from a first location of a tissue sample under conditions that release the plurality of oligonucleotides from the first location of the tissue sample;
b) collecting a plurality of oligonucleotides from a second location of the tissue sample under conditions that release the plurality of oligonucleotides from the second location of the tissue sample;
c) synthesizing a first plurality of DNA products by performing a synthesis reaction that uses the plurality of oligonucleotides collected in step (a) as templates and incorporates at least one nucleic acid sequence that identifies the first location of the tissue sample into each of the first plurality of DNA products;
d) synthesizing a second plurality of DNA products by performing a synthesis reaction that uses the plurality of oligonucleotides collected in step (b) as templates and incorporates at least one nucleic acid sequence that identifies the second location of the tissue sample into each of the second plurality of DNA products; and
e) identifying the first plurality of DNA products and the second plurality of DNA products synthesized in step (c) and step (d) by sequencing the first plurality of DNA products and the second plurality of DNA products, thereby spatially detecting the plurality of oligonucleotides collected from the first location of the tissue sample and the plurality of oligonucleotides collected from the second location of the tissue sample.
2. The method of claim 1 , wherein the tissue sample is immobilized onto a microscope slide.
3. The method of claim 2 , wherein the microscope slide comprises a plurality of primers immobilized on the microscope slide.
4. The method of claim 3 , wherein the plurality of primers is immobilized on the microscope slide at their 5′ ends.
5. The method of claim 4 , wherein each of step (c) and step (d) comprises performing a solid-phase amplification reaction, wherein the solid-phase amplification reaction is carried out on the microscope slide using the plurality of primers immobilized on the microscope slide.
6. The method of claim 1 , wherein the sequencing step is performed using a next generation sequencing reaction.
7. The method of claim 1 , further comprises amplifying a library using the first plurality of DNA products and the second plurality of DNA products as templates.
8. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the first location of the tissue sample comprise at least one unique molecular identifier.
9. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the second location of the tissue sample comprise at least one unique molecular identifier.
10. The method of claim 1 , wherein the first plurality of DNA products further comprises at least one amplification primer binding site.
11. The method of claim 1 , wherein the second plurality of DNA products further comprises at least one amplification primer binding site.
12. The method of claim 1 , wherein steps (a) and (b) are performed simultaneously.
13. The method of claim 1 , wherein steps (c) and (d) are performed simultaneously.