Single cell analysis

Abstract

The present invention concerns processes for barcoding nucleic acids from single cells and processes for genotyping ingle cells having a phenotype of interest.

1 - 7 . (canceled) 
     
     
         8 . A microfluidic process for barcoding cell nucleic acids, said method comprising:
 providing a microfluidic device comprising a chip comprising at least one microfluidic channel with at least one inlet, and a plurality of reservoirs,   injecting into one of said at least one inlet of the microfluidic channel a first carrier fluid comprising a plurality of droplets of a first type dispersed in the first carrier fluid, wherein the droplets of the first type are either cell droplets or reagents droplets, wherein at least some of the reagents droplets comprise an enzyme for barcoding cellular nucleic acids and at least one oligonucleotide, and wherein at least some of the cell droplets comprise one single cell, wherein said single cell comprises single cell nucleic acids,   migrating at least one droplet of the first type among the plurality of droplets into one reservoir of said plurality of reservoirs,   injecting into one of at least one inlet of the microfluidic channel, a second carrier fluid comprising a plurality of droplets of a second type dispersed in the second carrier fluid, wherein the droplets of the second type are either cell droplets or reagents droplets, wherein at least some of the reagents droplets comprise an enzyme for barcoding cellular nucleic acids and at least one oligonucleotide, wherein said at least one oligonucleotide comprises at least one barcode sequence, wherein at least some of the cell droplets comprise one single cell, wherein said single cell comprises cell nucleic acids, and wherein the droplets of the second type are reagents droplets when the droplets of the first type are cell droplets or the droplets of the second type are cell droplets when the droplets of the first type are reagents droplets,   contacting at least one part of at least one droplet of the second type with one reservoir of said plurality of reservoirs,   for a plurality of reservoirs, fusing, in or at the edge of each reservoir, said at least one droplet of the first type with said at least one droplet of the second type, thereby resulting in a fused droplet,   and further comprising the steps of:
 a) contacting, for each fused droplet, the at least some of the cell nucleic acids with the at least one oligonucleotide in said fused droplet, 
 b) barcoding, in each fused droplet, at least some of the cell nucleic acids present in said fused droplet, thereby resulting in cell barcoded nucleic acid, and 
 c) associating at least one barcode sequence to the cell barcoded nucleic acid obtained in step b), 
   or   further comprising the steps of:
 a) contacting, for each fused droplet, at least some of cell nucleic acids with the at least one oligonucleotide in said fused droplet, wherein said at least one oligonucleotide comprises at least one barcode sequence, 
 b) barcoding, in each fused droplet, at least some of the cell nucleic acids present in said fused droplet, thereby resulting in barcoded cell nucleic acid. 
   
     
     
         9 . The microfluidic process according to  claim 8 , wherein the reagents-droplets comprises reverse transcriptase, nuclease, polymerase, or ligase enzymes. 
     
     
         10 . The microfluidic process according to  claim 8 , wherein the enzyme-droplets further comprise a polymer. 
     
     
         11 . The microfluidic process according to  claim 8 , wherein the plurality of droplets of the first type have an average volume from 1 pL to 3000 pL. 
     
     
         12 . The microfluidic process according to  claim 8 , wherein the plurality of droplets of the second type have an average volume from 10 pL to 5000 pL. 
     
     
         13 . The microfluidic process according to  claim 8 , wherein migrating at least one droplet of the first type results in an occupancy of 90% to 100% of the total number of reservoirs and/or results in a capturing efficiency of 80% to 100% of the injected droplets of the first type. 
     
     
         14 . The microfluidic process according to  claim 8 , wherein the at least one part of at least one droplet of the second type refers to 90% to 100% of the total volume of the at least one droplet of the second type. 
     
     
         15 . The microfluidic process according to  claim 8 , wherein the fusion step results in a fusion efficiency of 90% to 100% between the droplets of the first type and the droplets of the second type. 
     
     
         16 . The microfluidic process according to  claim 8 , wherein a phenotypic assay is performed on said cell(s) at any step before, during or after the barcoding of said cell(s) nucleic acid. 
     
     
         17 . The microfluidic process according to  claim 8 , wherein at least some of the reagents-droplets comprises at least one particle of a first type to which the at least one oligonucleotide is bound. 
     
     
         18 . A device for barcoding cell nucleic acids, said device comprising:
 a microfluidic device comprising a chip comprising at least one microfluidic channel with at least one inlet, and a plurality of reservoirs,   a fluidic device capable of performing each or individual operations:
 injecting into one of said at least one inlet of the microfluidic channel a first carrier fluid comprising a plurality of droplets of a first type dispersed in the first carrier fluid, wherein the droplets of the first type are either cell droplets or reagents droplets, wherein at least some of the reagents droplets comprise an enzyme for barcoding cellular nucleic acids and at least one oligonucleotide, and wherein at least some of the cell droplets comprise one single cell, wherein said single cell comprises cell nucleic acids, 
 controlling a first migration step, wherein at least one droplet of the first type among the plurality of droplets is moved into one reservoir of said plurality of reservoirs, 
 injecting into one of said at least one inlet of the microfluidic channel, a second carrier fluid comprising a plurality of droplets of a second type dispersed in the second carrier fluid, wherein the droplets of the second type are either cell droplets or reagents droplets, wherein at least some of the reagents droplets comprise an enzyme for barcoding cellular nucleic acids and at least one oligonucleotide, wherein said at least one oligonucleotide comprises at least one barcode sequence, and wherein at least some of the cell droplets comprise one single cell, wherein said single cell comprises cell nucleic acids, and wherein the droplets of the second type are reagents droplets when the droplets of the first type are cell droplets or the droplets of the second type are cell droplets when the droplets of the first type are reagents droplets, 
 controlling a second migration step for a plurality of reservoirs, wherein, at least one part of at least one droplet of the second type is in contact with one reservoir of said plurality of reservoirs, 
   a fusion device triggering fusion for a plurality of reservoirs, in or at the edge of each reservoir, of said at least one droplet of the first type with said at least one droplet of the second type, thereby resulting in a fused droplet,   and further comprising one or several devices for:
 a) contacting, for each fused droplet, at least some of the cell nucleic acids with the at least one oligonucleotide in said fused droplet, 
 b) barcoding, in each fused droplet, at least some of the cell nucleic acids present in said fused droplet, thereby resulting in cell barcoded nucleic acid, and 
 c) linking at least one barcode sequence to the cell barcoded nucleic acid obtained in step b), 
   or   further comprising one or several devices for:
 a) contacting, for each fused droplet, at least some of cell nucleic acids with the at least one oligonucleotide in said fused droplet, wherein said at least one oligonucleotide comprises at least one barcode sequence, 
 b) barcoding, in each fused droplet, at least some of the cell nucleic acids present in said fused droplet, thereby resulting in barcoded cell nucleic acid. 
   
     
     
         19 . The microfluidic process according to  claim 8 , wherein the at least one part of the at least one droplet of the second type refers to 10% to 30% of the total volume of the at least one droplet of the second type.