MULTI-OMIC ANALYSIS IN MONODISPERSE DROPLETS
This disclosure provides methods and systems for single-cell, multi-omic 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. Nucleic-acid-tagged antibody conjugates are used for simultaneous proteomic analysis along with the gene expression profiling.
1 . A method for single cell analysis, the method comprising:
incubating a plurality of nucleic-acid-labelled, target-specific antibodies with a plurality of target cells to promote binding of the nucleic-acid-labelled, target-specific antibodies to target proteins expressed by the target cells;
washing the incubated target cells to remove unbound nucleic-acid-labelled, target-specific antibodies;
combining in a first fluid template particles and the washed target cells;
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;
amplifying and sequencing nucleic acid labels from the nucleic-acid-labelled, target-specific antibodies to identify target proteins expressed by the target cells.
2 . The method of claim 1 further comprising quantifying the target proteins expressed by the target cells.
3 . The method of claim 2 wherein the nucleic acid labels comprise a unique molecular identifier sequence.
4 . The method of claim 2 wherein the nucleic acid labels comprise a PCR handle.
5 . The method of claim 1 further comprising lysing each of the single target cells contained within the monodisperse droplets to release a plurality of distinct mRNA molecules; and
quantifying the plurality of distinct mRNA molecules.
6 . The method of claim 5 wherein the nucleic acid labels comprise a capture portion.
7 . The method of claim 6 wherein the capture portion comprises a poly A sequence.
8 . The method of claim 5 , further comprising generating an expression profile for each of the single target cells after quantifying the plurality of distinct mRNA molecules.
9 . The method of claim 5 , further comprising reverse transcribing the plurality of distinct mRNA molecules inside the droplets.
10 . The method of claim 1 , wherein the first fluid is an aqueous fluid.
11 . The method of claim 10 , wherein the second fluid comprises an oil.
12 . The method of claim 11 , wherein shearing the fluids comprises one of using a vortexer or pipetting.
13 . The method of claim 5 , wherein the template particles further comprise one or more compartments.
14 . The method of claim 13 , 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.
15 . The method of claim 14 , wherein the nucleic acid synthesis reagent comprises a polymerase.
16 . The method of claim 14 , wherein the reagent is released from the one or more compartments in response to an external stimulus.
17 . The method of claim 6 , wherein the template particles comprise a plurality of capture probes comprising:
a universal primer sequence;
at least one barcode; and
a capture sequence that hybridizes to one or more of the plurality of distinct mRNA.
18 . The method of claim 17 , wherein the capture sequence is selected from one of a poly T nucleotide sequence, a gene-specific nucleotide sequence, or a random nucleotide sequence.
19 . The method of claim 17 , wherein the capture sequence of one or more of the capture probes hybridizes to the capture portion of one or more of the nucleic acid labels.
20 . The method of claim 17 , wherein mRNA attached to the template particle by the capture probes is reverse transcribed to generate a first strand comprising cDNA and the barcode sequence.
21 . The method of claim 20 , further comprising amplifying the first strand by PCR to generate amplicons of the first strand DNA and the nucleic acid labels.