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 - 21 . (canceled)
22 . A method comprising:
combining template particles with cells in a first fluid wherein the template particles are linked to copies of a first capture probe comprising a poly-T sequence and a second capture probe comprising a template-switching segment;
generating droplets of the first fluid, in a second immiscible fluid, wherein at least one droplet contains a single template particle and a single target cell;
breaking the droplets;
lysing the target cell to release a plurality of distinct mRNA molecules in the droplet;
capturing at least one mRNA molecule in the droplet with the poly-T sequence of the first capture probes of the single template particle;
reverse transcribing the mRNA molecule to yield a cDNA using a reverse transcriptase that adds additional bases to a 3′ end the cDNA;
annealing the additional bases of the cDNA to the template-switching segment of the second capture probe; and
copying, by the reverse transcriptase, the annealed second capture probe into the cDNA.
23 . The method of claim 22 , wherein the copying step leaves the cDNA linked to bead, the cDNA comprising first and second universal primer binding sites, at least one cell barcode, and at least one unique molecular identifier (UMI).
24 . The method of claim 22 , wherein the template-switching segment comprises a plurality of ribo-Gs.
25 . The method of claim 22 , wherein the additional bases added by the reverse transcriptase comprise CCC.
26 . The method of claim 22 , wherein the second capture probe comprises, from 5′ to 3′ end: a covalent bond with the template particle; a first universal primer nucleotide sequence, a first barcode, and a capture sequence that includes the poly-T nucleotide sequence.
27 . The method of claim 22 , wherein the second capture probe comprises one or more of the template switching segment, a UMI, a second barcode, and a second universal primer nucleotide sequence.
28 . The method of claim 22 , wherein the generating step comprises adding the second fluid to the first fluid in a tube, and vortexing the tube to shear the first and second fluids, causing the droplets to form simultaneously and monodisperse.
29 . The method of claim 22 , further comprising quantifying the plurality of distinct mRNA molecules and optionally generating an expression profile for the cells after quantifying the plurality of distinct mRNA molecules.
30 . The method of claim 29 , wherein the first fluid is an aqueous fluid and the second fluid comprises an oil.
31 . The method of claim 30 , wherein the template particles further comprise one or more compartments.
32 . The method of claim 31 , wherein the one or more compartments contain a reagent selected from a group comprising a lytic reagent, a nucleic acid synthesis reagent, or combination thereof.
33 . The method of claim 32 , wherein the nucleic acid synthesis reagent comprises a murine leukemia virus reverse transcriptase.
34 . The method of claim 22 , further comprising amplifying the cDNA by PCR to generate amplicons.
35 . The method of claim 34 , wherein amplicons are sequencing and counted using UMIs to deduplicated the amplicons.
36 . The method of claim 34 , wherein said amplifying step comprises adding primer binding sites comprising capture probes at 3′ and 5′ ends of each cDNA.
37 . The method of claim 34 , wherein the amplifying step comprises using a universal primer site on the second capture probe and sets of gene-specific primers.
38 . The method of claim 22 , wherein the breaking step occurs after the capturing step.
39 . The method of claim 22 , wherein the breaking step occurs after the reverse transcribing step.