Methods and systems for single cell gene profiling
This disclosure provides methods and systems for single-cell analysis, including single-cell transcriptome analysis, of target cells without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target cell from a population of cells in an encapsulation, derive a plurality of distinct mRNA molecules from the single target cell, and quantify the distinct mRNA molecules to generate an expression profile.
1. A method for single cell analysis, the method comprising:
combining template particles with target cells in a first fluid, wherein each template particle is bound to first capture probes comprising capture sequences and second capture probes comprising template-switching oligos (TSOs);
adding a second fluid to the first fluid;
shearing the fluids to generate a plurality of monodisperse droplets simultaneously that contain a single one of the template particles and a single one of the target cells;
lysing each of the single target cells contained within the monodisperse droplets to release a plurality of distinct mRNA molecules;
capturing the plurality of distinct mRNA molecules with the first capture probes;
extending the first capture probes with reverse transcriptase to form nascent first strand cDNAs;
annealing the nascent first strand cDNAs to the TSOs and copying the TSOs to generate cDNAs that are bound to the template particles and contain copies of the plurality of distinct mRNA molecules and the TSOs; and
analyzing the cDNAs to quantify the plurality of distinct mRNA molecules.
2. The method of claim 1 , further comprising generating an expression profile for each of the single target cells after quantifying the plurality of distinct mRNA molecules.
3. The method of claim 1 , wherein shearing the fluids comprises one of using a vortexer or pipetting.
4. The method of claim 3 , wherein the template particles comprise features that facilitate capture of the single cells and one or more encapsulated internal compartments that contain reagents.
5. The method of claim 4 , wherein the one or more encapsulated internal compartments contain a reagent selected from a group comprising a lytic reagent, a nucleic acid synthesis reagent, or combination thereof.
6. The method of claim 5 , wherein the nucleic acid synthesis reagent comprises a polymerase.
7. The method of claim 4 , wherein the features that capture the single cells include flat surfaces, craters, or grooves.
8. The method of claim 1 , wherein the first of capture probes are oligonucleotides, each comprising:
a 5′ terminal acrydite group;
a universal primer sequence;
at least one barcode; and
one of the capture sequences.
9. The method of claim 8 , wherein the capture sequence is selected from one of a poly T nucleotide sequence, a gene-specific nucleotide sequence, or a random nucleotide sequence.
10. The method of claim 1 , wherein each of the plurality of distinct mRNA molecules attaches to the template particle by hybridizing to a poly T nucleotide sequence upon release from the single target cells.
11. The method of claim 10 , further comprising releasing the cDNAs from the template particles and amplifying the cDNAs by PCR to generate amplicons.
12. The method of claim 10 , wherein the mRNA attached to the template particle is reverse transcribed using the TSOs to add a common 5′ sequence to the cDNAs.
13. The method of claim 11 , wherein quantifying the plurality of distinct mRNA molecules comprises sequencing the amplicons.
14. The method of claim 11 , wherein at least one of the target cells 1s a cancer cell.
15. A kit for single cell profiling, the kit comprising:
a tube containing template particles, wherein each template particle is bound to first capture probes comprising capture sequences and second capture probes comprising template switching oligos (TSOs), features that facilitate capture of single cells onto a surface of the template particle, and one or more encapsulated internal compartment containing a reagent.
16. The kit of claim 15 , wherein the reagent is reverse transcriptase.
17. The kit of claim 15 , wherein the features that facilitate capture include craters or flat surfaces on generally spherical surfaces of the template particles.