Simultaneous spatio-temporal measurement of gene expression and cellular activity
Provided herein are methods for simultaneous spatio-temporal measurement of gene expression and cellular activity.
1. A method for identifying location and abundance of an analyte in a plurality of live cells, the method comprising:
(a) contacting the plurality of live cells with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain that binds to a sequence present in the analyte;
(b) recording a cellular activity or an intracellular gene expression of a nucleic acid in the plurality of live cells, wherein the plurality of live cells is located within a perfusion chamber comprising a gasket and the substrate, wherein the gasket comprises (i) a plurality of apertures, (ii) a plurality of input channels being fluidly connected to the plurality of apertures, respectively, and (iii) a plurality of output channels being fluidly connected to the plurality of apertures, respectively;
(c) hybridizing the analyte to the capture domain of the capture probe;
(d) extending the capture probe using the analyte as a template, thereby generating an extended capture probe;
(e) amplifying the extended capture probe to produce a plurality of extended capture probes; and
(f) sequencing (i) the spatial barcode, or a complement thereof, and (ii) all or part of the sequence of the analyte, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location and abundance of the analyte in the plurality of live cells,
wherein step (b) occurs before steps (c), (d), (e), and (f).
2. The method of claim 1 , further comprising mounting the gasket onto the substrate,
wherein the substrate comprises a plurality of substrate regions, wherein a substrate region of the plurality of substrate regions comprises the capture probe comprising (i) the spatial barcode and (ii) the capture domain that binds to the sequence present in the analyte, and
wherein the plurality of apertures of the gasket are aligned with the plurality of substrate regions of the substrate when the gasket is mounted onto the substrate.
3. The method of claim 2 , further comprising mounting a cover onto the gasket,
wherein the cover comprises an inlet and an outlet, the inlet being fluidly connected to the plurality of input channels when the cover is mounted onto the gasket, the outlet being fluidly connected to the plurality of output channels when the cover is mounted onto the gasket, and
wherein the perfusion chamber further comprises the cover that is mounted onto the gasket.
4. The method of claim 1 , further comprising perfusing a test compound through the perfusion chamber.
5. The method of claim 4 , wherein the test compound is a drug.
6. The method of claim 4 , wherein the perfusing step occurs prior to the recording step, or wherein the perfusing step and the recording step occur at substantially the same time.
7. The method of claim 1 , further comprising culturing the plurality of live cells in the perfusion chamber before the recording step.
8. The method of claim 7 , wherein the plurality of live cells is cultured in a culture medium in the perfusion chamber.
9. The method of claim 8 , wherein the culture medium is supplemented with oxygen.
10. The method of claim 8 , wherein the culture medium further comprises a blocking reagent, or wherein the plurality of live cells is treated with the blocking reagent.
11. The method of claim 1 , wherein the recording step comprises optical recording.
12. The method of claim 11 , wherein the optical recording comprises contacting the plurality of live cells with a chemical dye or an indicator.
13. The method of claim 12 , wherein the chemical dye or the indicator comprises a fluorophore.
14. The method of claim 11 , wherein the optical recording is achieved by in situ hybridization or fluorescence resonance energy transfer.
15. The method of claim 11 , wherein the optical recording comprises hybridization of a plurality of optically-labelled probes to (a) a protein, a lipid, a nucleic acid, or a combination thereof associated with the cellular activity; or (b) the nucleic acid associated with the intracellular gene expression.
16. The method of claim 15 , wherein an optically-labelled probe of the plurality of optically-labelled probes is a peptide nucleic acid probe labelled with a fluorophore.
17. The method of claim 1 , wherein the plurality of live cells is a cell culture sample or a tissue sample.
18. The method of claim 17 , wherein the tissue sample is a live tissue section, an organoid sample, or a spheroid culture sample.
19. The method of claim 17 , wherein the cell culture sample is a primary cell culture sample comprising cells from a fresh tissue.
20. The method of claim 1 , wherein the substrate is a coverslip comprising plastic, metal, or glass.
21. The method of claim 1 , wherein the plurality of live cells is stained by immunohistochemistry or immunofluorescence before the recording step.
22. The method of claim 1 , wherein the plurality of live cells is stained using a detectable label, wherein the detectable label is Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, or Periodic Acid Schiff (PAS).
23. The method of claim 1 , wherein the plurality of live cells is imaged using bright field imaging.
24. The method of claim 1 , wherein the plurality of live cells is permeabilized with a permeabilization agent selected from an organic solvent, a cross-linking agent, a detergent, and an enzyme, or a combination thereof, wherein the plurality of live cells is permeabilized after step (b) and before step (c).
25. The method of claim 1 , wherein the extending step comprises extending the capture probe at the 3′ end.
26. The method of claim 1 , wherein the plurality of live cells is removed after the amplifying step.
27. The method of claim 1 , wherein the amplifying step comprises amplifying (i) all or part of the sequence of the analyte bound to the capture domain, or a complement thereof, and (ii) the spatial barcode, or a complement thereof.
28. The method of claim 1 , wherein the identifying further comprises correlating the recording of the intracellular gene expression of the nucleic acid in the plurality of live cells to the location and abundance of the analyte in the plurality of live cells.