DECENTRALIZED WORKFLOWS FOR SINGLE CELL ANALYSIS
This disclosure provides a decentralized workflow for analyzing single cell gene expression. The workflow makes use of pre-templated instant partitions to segregate cells into separate compartments to individually capture and barcode RNA from single cells in a massively parallel single tube format. The workflow includes steps for processing the RNA from the single cells for sequencing. Separate portions of the decentralized workflow are performed by a research lab and a core facility, allowing increased flexibility in time and location of protocol steps.
1 . A decentralized method for single cell analysis, the method comprising steps:
(a) partitioning a mixture to generate a plurality of partitions, simultaneously, inside of a vessel, wherein the partitions contain a single cell and a template particle that are isolated from the mixture comprising:
an aqueous solution;
template particles comprising barcoded oligos;
cells; and
an oil;
(b) lysing the cells inside the partitions and capturing mRNA of single cells with barcoded oligos of the template particles;
(c) copying the mRNA of the single cells into barcoded cDNA;
(d) amplifying the barcoded cDNA to create amplicons;
(e) preparing sequencing libraries from the amplicons;
(f) sequencing the libraries to produce single cell gene expression data;
wherein, step (a) is performed at a research lab and step (f) is performed at a core facility.
2 . The method of claim 1 , wherein steps (e) and (f) are performed at the core facility.
3 . The method of claim 1 , wherein steps (d), (e), and (f), are performed at the core facility.
4 . The method of claim 1 , wherein steps (c), (d), (e), and (f), are performed at the core facility.
5 . The method of claim 1 , wherein steps (b), (c), (d), (e), and (f), are performed at the core facility.
6 . The method of claim 1 , wherein partitioning comprises vortexing the mixture to shear the aqueous solution into droplets surrounded by the oil.
7 . The method of claim 6 , wherein the droplets are formed around the template particles.
8 . The method of claim 6 , wherein the oil comprises a surfactant that stabilizes the partitions.
9 . The method of claim 8 , further comprising mailing the vessel comprising the partitions to the core facility for processing.
10 . The method of claim 1 , wherein the cDNA is linked to the template particles via the barcoded oligos.
11 . The method of claim 10 , wherein the cDNA is mailed to the core facility.
12 . The method of claim 11 , wherein no PCR steps are performed by the research lab.
13 . The method of claim 1 , wherein the amplicons are generated by the research lab.
14 . The method of claim 13 , wherein the amplicons are mailed to the core facility.
15 . The method of claim 1 , wherein at least one of the steps is performed by the research lab at a remote location.
16 . The method of claim 1 , wherein the cells comprise an infectious agent.
17 . The method of claim 16 , wherein the research lab, but not the core facility, is qualified to handle the infectious agent.
18 . The method of claim 17 , wherein the infectious agent is neutralized by the research lab before any cell material is transferred to the core facility.
19 . The method of claim 1 , wherein the research lab and the core facility perform the method steps with a sample preparation kit comprising reagents and instructions that is provided by a third party.
20 . The method of claim 19 , wherein the kits are specific to the steps performed at the research lab and core facility.