Core-shell gel particles for separating and analyzing RNA and DNA from a cell

Abstract

Provided herein are compositions, systems, kits, and methods for separating and analyzing RNA / cDNA from genomic DNA molecules using core-shell gel particles (e.g., such that separate barcoded sequencing libraries can be generated from each). In certain embodiments, the core-shell gel particles comprise an outer gel shell, and an interior region surrounded by said shell that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) at least one cell, and D) a plurality of capture beads comprising capture probes configured to hybridize to RNA from the cell, and optionally configured to hybridize to genomic DNA and / or bind target proteins.

Description

Abstract This invention provides compositions, systems, kits, and methods for isolating and analyzing RNA / complementary DNA (cDNA) from genomic DNA molecules, using core-shell gel particles (e.g., enabling the generation of individual barcoded sequencing libraries from which). In some embodiments, the core-shell gel particles comprise an outer gel shell and an internal region surrounded by the shell and comprising: A) an internal solution at least partially aqueous; B) an optional inner gel core; C) at least one cell; and D) a plurality of capture microspheres containing capture probes configured to hybridize with RNA from the cell and optionally configured to hybridize with genomic DNA and / or bind target proteins.

Claims

CLAIMSWc claim:

1. A method comprising: a) generating an emulsion comprising a plurality of aqueous droplets in an oil phase such that at least some of said aqueous droplets are positive droplets that each comprise: i) an aqueous solution; ii) a gelling agent, iii) at least one cell, and iv) optionally a plurality of capture beads comprising: A) capture probes configured to hybridize to RNA from said cell, and optionally B) DNA capture sequences configured to hybridize to DNA from said cell; and optionally C) protein binding agents; b) treating said positive droplets in said emulsion such that said gelling agent gels in a plurality of said positive droplets thereby forming a plurality of core-shell gel particles that are present in said oil phase and that each comprise: i) an outer shell, and ii) an interior region, surrounded by said shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) said at least one cell, and D) optionally at least some of said capture beads, wherein said treating optionally comprises cooling said positive droplets, and c) adding a lysing agent solution to said emulsion such that at least pail of said lysing solution agent migrates through said outer shell into said interior region and lyses said at least one cell releasing RNA and DNA molecules in each of said plurality of core-shell gel particles.

2. The method of claim 1, wherein said plurality of capture beads are present but without said DNA capture sequences, and the method further comprises: d) incubating said emulsion such that in each of said plurality of core-shell gel particles: A) at least some of said RNA molecules specifically hybridize to said capture probes thereby generating RNA-bound capture beads, and B) said DNA molecules are present in said interior solution and do not specifically hybridize to said capture probes, and do not migrate out of through said outer shell; andoptionally e) adding reverse-tran scription reagents to said emulsion to generate cDNA from said RNA bound to said RNA-bound capture beads.

3. The method of claim 2, further comprising: e) transferring at least some of said plurality of core-shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample and a second sample, wherein said first sample comprises said RNA-bound capture beads and is free or substantially free of said DNA molecules, and wherein said second sample comprises all or substantially all of said DNA molecules and is free or substantially free of said RNA molecules.

4. The method of claim 3, wherein said capture probes are attached to said capture beads via a reversible linker, and further comprising: g) treating said first sample such that said RNA molecules are reverse transcribed into cDNA either on said capture beads or after having been cleaved from said capture beads via said reversible linker.

5. The method of claim 4, further comprising: h) generating a sequencing library from said cDNA by attaching adapters to said cDNA or amplicons thereof and amplifying said cDNA and / or said amplicons using primers, wherein optionally said capture probes and / or said adapter comprise a barcode sequence, and i) sequencing said sequencing library to generate a plurality of RNA / cDNA sequencing reads.

6. The method of claim 3, further comprising: h) generating a sequencing library from said DNA molecules in said second sample, by attaching adapters to said DNA or amplicons thereof and amplifying said DNA molecules and / or said amplicons using primers, wherein optionally said adapters comprise a barcode sequence, and i) sequencing said sequencing library to generate a plurality of DNA sequencing reads.

7. The method of claim 1, wherein said plurality of capture beads are present with said DNA capture sequences, and the method further comprises: d) incubating said emulsion such that in each of said plurality of core-shell gel particles: A) at least some of said RNA molecules specifically hybridize to said capture probes, and B) at least some of said DNA moleculesspecifically or non-specifically hybridize to said DNA capture sequences; thereby generating RNA + DNA bound capture beads.

8. The method of claim 7, further comprise: e) transferring at least some of said plurality of core- shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified RNA + DNA bound capture beads.

9. The method of claim 8, wherein said capture probes are attached to said capture beads via a reversible linker, and further comprising: g) at least one of the following:A) cleaving said RNA molecules from said RNA + DNA capture beads via said reversible linker to generate free RNA and DNA-capture beads, and separating said free RNA into a second sample and said DNA-capture beads into a third sample, and reverse transcribing said free RNA in said second sample into cDNA; orB) reverse transcribing said RNA on said RNA + DNA capture molecules into cDNA molecules, and cleaving said RNA and cDNA molecules from said RNA + DNA capture beads via said reversible linker to generate free RNA, free cDNA, and DNA-capture beads, and separating said free RNA and free cDNA into a second sample and said DNA-capture beads into a third sample.

10. The method of claim 9, further comprising: h) generating a first sequencing library from said cDNA from said second sample by attaching adapters to said cDNA or to amplicons thereof, and amplifying said cDNA and / or amplicons thereof using primers, wherein optionally said capture probes and / or said adapters each comprise a barcode sequence, and i) sequencing said first sequencing library to generate a plurality of RNA / cDNA sequencing reads.

11. The method of 9, further comprising: h) generating a second sequencing library from said DNA molecules in said third sample by attaching adapters to said DNA molecules or to amplicons thereof, and amplifying said DNA molecules and / or amplicons using primers, wherein optionally said capture sequences and / or said adapters each comprise a barcode sequence and i) sequencing said second sequencing library to generate a plurality of DNA sequencing reads.

12. The method of claim 1, wherein said plurality of capture beads arc not present, and the method further comprises: d) incubating said emulsion such that substantially all of said RNA molecules in each of said plurality of core-shell particles migrate out of said interior region through said outer shell and such that substantially all of said DNA molecules remain inside said outer shell in each of said core-shell particles.

13. The method of claim 12, further comprising: e) transferring at least some of said plurality of core-shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified DNA molecules.

14. The method of claim 13, further comprising: f) generating a sequencing library from said DNA molecules in said first sample by attaching adapters to said DNA molecules or to amplicons thereof, and amplifying said DNA molecules and / or amplicons using primers, wherein optionally said adapters each comprise a barcode sequence and i) sequencing said sequencing library to generate a plurality of DNA sequencing reads.

15. The method of claim 1, wherein said at least one cell in each of said plurality of coreshell gel particles is one and only one cell.

16. The method of claim 1, wherein said generating said emulsion comprises either:1) combining an aqueous phase with said oil phase in bulk solution, wherein said aqueous phase comprises: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads, or2) the following steps: i) forming a first precursor emulsion comprising a plurality of first precursor aqueous droplets in an oil phase that each contain a precursor core-shell gel particle, ii) combining said plurality of first precursor aqueous droplets with a solution comprising cells to generate a second precursor emulsion comprising second precursoraqueous droplets in an oil phase that each contain at least one cell and a precursor coreshell gel particle, iii) heating said second precursor emulsion such that said precursor core-shell gel particle in each of said second precursor aqueous droplets melts forming liquid gel precursor, thereby forming said plurality of aqueous droplets recited in step a).

17. The method of claim 1, wherein said generating said emulsion comprises combining a first aqueous phase with said oil phase using a microfluidic droplet maker, wherein; I) said first aqueous phase comprises one or more of the following: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads, or II) wherein a second aqueous phase is combined with said first aqueous phase and said oil phase to generate said emulsion, and wherein said second aqueous phase comprises one or more of the following: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads.

18. The method of claim 17, wherein said microfluidic droplet maker comprises a co-flow droplet maker, and optionally comprises: a How focusing component, a T-junction, a step emulsification component, and / or a jet triggered droplet generator.

19. The method of claim 1, wherein said gelling agent comprises chitosan.

20. The method of claim 17, wherein said microfluidic droplet maker employs particle templated emulsification or geometrically mediated breakup in a hierarchical array and optionally without requiring the substantial control of flow rate.

21. The method of claim 1, wherein said positive droplets further comprise cell media.

22. The method of claim 1, wherein said positive droplets comprise at least to cells, or only two cells.

23. The method of claim 1 , wherein said positive droplets have a diameter of from about 1 pm to 1000 pm.

24. The method of claim 1, wherein said lysing agent solution comprises a lysing agent, wherein said lysing agent is optionally a protease.

25. The method of claim 1, wherein said lysing agent solution comprises a detergent or other chemical that overcomes inhibition and optionally facilitate barcoding of nucleic acids and / or micelle transport.

26. The method of any of claims 5, 6, 10, 11, and 14 wherein said amplifying is performed by PCR or multiple displacement amplification (MDA).

27. The method of any of claims 5, 6, 10, 11, and 14, wherein said sequencing is performed by a method selected from: Ab-seq, CITE-seq, sequencing by synthesis, and next generation sequencing.

28. The method of any of claims 1-27, wherein said plurality of capture beads are present and comprise said protein binding agents.

29. The method of claim 28, wherein said protein binding agents bind target proteins from said at least one cell, and optionally wherein said target proteins are purified into a purified protein sample.

30. The method of claim 1, wherein said emulsion is a single emulsion or double emulsion.

31. The method of claim 30, further comprising: sorting said plurality of core-shell gel particles based on the presence or absence of a signal or detectable molecule from each of said plurality of core-shell gel particles.

32. The method of claim 31 , wherein said sorting is based on the presence or absence of said detectable molecule arc particular barcoded or unbarcodcd DNA or mRNA molecules.

33. A composition, system, or kit comprising: a) a plurality of core-shell gel particles that each comprise: i) an outer gel shell, and ii) an interior region, surrounded by said shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) said at least one cell, and D) a plurality of capture beads comprising: I) capture probes configured to hybridize to RNA from said cell, and optionally II) DNA capture sequences configured to hybridize to DNA from said cell; and optionally III) protein binding agents; and b) optionally a lysing agent solution.

34. The composition, system, or kit of claim 33, further comprising an aqueous solution or an oil carrier phase, and wherein said plurality of core-shell gel particles are present in said aqueous solution or said oil carrier phase.

35. The method, composition, system, or kit of any of the above claims, wherein the RNA comprising mRNA.