Compositions, methods, and systems for single cell barcoding and sequencing

Abstract

This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), the plurality of droplets generated from an aqueous mixture comprising a set of single cells and a set of functionalized particles configured for a single cell assay. Upon generation, the plurality of droplets can be stabilized in position within a region of the collecting container, thereby providing a single-tube workflow for single cell analyses. Further, compositions implemented are structured to allow for overloading of partitions with functionalized particles, such that partitioned single-cells are co-localized with a subset of functionalized particles in a manner that allows for discernable tagging and downstream analyses.

What is claimed is: 
     
         1 . A method comprising:
 generating an emulsion within a collecting container, the emulsion comprising a plurality of droplets generated from an aqueous mixture, wherein droplets of the plurality of droplets are individually separated by an oil within the collecting container,   wherein the plurality of droplets comprises 1 million droplets generated upon spinning the oil within the collecting container for a duration of 2 minutes, and   wherein the aqueous mixture comprises a set of biological targets.   
     
     
         2 . The method of  claim 1 , wherein the set of biological targets comprises a set of single cells, and wherein the aqueous mixture further comprises: (i) a set of functionalized particles configured for a single cell assay and (ii) a lysis reagent. 
     
     
         3 . The method of  claim 2 , wherein the set of functionalized particles comprises polyacrylamide particles coupled to a set of molecules comprising (i) capture probes configured to capture mRNA molecules of the set of single cells and (ii) barcode segments unique to each functionalized particle of the set of functionalized particles. 
     
     
         4 . The method of  claim 2 , further comprising transmitting heat to the plurality of droplets, thereby lysing single cells of the set of single cells. 
     
     
         5 . The method of  claim 2 , further comprising capturing mRNA molecules of the set of single cells at the set of functionalized particles and performing reverse transcription of the mRNA molecules into cDNA molecules. 
     
     
         6 . The method of  claim 5 , further comprising generating a single cell expression library for the set of single cells upon sequencing the cDNA molecules. 
     
     
         7 . The method of  claim 2 , wherein the set of single cells comprises breast cancer cells. 
     
     
         8 . The method of  claim 2 , wherein the set of single cells comprises blood cells and immune cells. 
     
     
         9 . The method of  claim 1 , wherein the plurality of droplets is monodisperse. 
     
     
         10 . The method of  claim 1 , wherein each droplet of the plurality of droplets comprises a diameter from 20-350 micrometers. 
     
     
         11 . A method comprising:
 generating an emulsion within a collecting container, the emulsion comprising a plurality of droplets generated from an aqueous mixture, wherein droplets of the plurality of droplets are individually separated by an oil within the collecting container,   wherein the plurality of droplets comprises 1 million droplets generated upon spinning the oil within the collecting container for a duration of 2 minutes,   wherein the plurality of droplets is monodisperse,   wherein the aqueous mixture comprises a set of single cells, a set of functionalized particles configured for a single cell assay, and a lysis reagent, and   wherein the set of functionalized particles comprise polyacrylamide particles with a set of molecules comprising capture probes configured to capture mRNA molecules of the set of single cells, and barcode segments unique to each of the set of functionalized particles;   transmitting heat to the plurality of droplets, thereby lysing single cells of the set of single cells;   capturing mRNA molecules of the set of single cells at the set of functionalized particles;   performing reverse transcription of the mRNA molecules into cDNA molecules; and   generating a single cell expression library for the set of single cells upon sequencing the cDNA molecules.   
     
     
         12 . The method of  claim 11 , wherein the set of single cells comprises cellular material derived from a tissue source. 
     
     
         13 . The method of  claim 11 , wherein the set of single cells comprises breast cancer cells. 
     
     
         14 . The method of  claim 11 , wherein the set of single cells comprises blood cells and immune cells. 
     
     
         15 . The method of  claim 11 , further comprising generating an analysis of disease pathology and progression, from the single cell expression library. 
     
     
         16 . The method of  claim 11 , wherein the capture probes comprise one of dT and dTVN. 
     
     
         17 . The method of  claim 11 , wherein generating the plurality of droplets comprises generating the plurality of droplets at a rate of at least 500,000 droplets per minute. 
     
     
         18 . The method of  claim 11 , wherein each droplet of a subset of the plurality of droplets comprises one single cell of the set of single cells, and one particle of the set of functionalized particles. 
     
     
         19 . The method of  claim 11 , wherein the set of single cells comprises stained cells. 
     
     
         20 . The method of  claim 11 , further comprising breaking the emulsion in coordination with said sequencing. 
     
     
         21 . The method of  claim 11 , wherein the set of molecules further comprises adapter segments and index segments for library preparation. 
     
     
         22 . The method of  claim 11 , wherein each droplet of the plurality of droplets comprises a diameter from 20-350 micrometers.