Microfluidic analysis of ligand induced cell expression

A method for determining the gene expression of a cell, comprising the steps:

• (a) providing a plurality of microfluidic compartments wherein at least 1% of said compartments form a subset in which each compartment is a droplet comprising:
• (i) a first cell,
• (ii) one second cell or one cell-free expression system, the second cell or the cell-free expression system expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on a surface of the first cell, and
• (iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and mRNA and/or cDNA of the one second cell or the one cell-free expression system,
• (b) lysing the cells comprised in the compartments,
• (c) reverse transcribing mRNA released from the cells to cDNA,
• (d) amplifying the cDNA,
• (e) determining the sequence and optionally the respective amount of the cDNA, and
• (f) selecting sequences comprising the same barcode sequence, wherein the following sequences are excluded:
• sequences derived from a second cell or a cell-free expression system,
• sequences comprising a unique barcode sequence that is not associated in other sequences with a nucleotide sequence encoding for a polypeptide ligand expressed by a second cell or a cell-free expression system,
• sequences comprising a unique barcode sequence that is associated with a nucleotide sequence encoding for more than one polypeptide ligand expressed by a second cell or a cell-free expression system,
• wherein step (c) or step (d) is carried out with a barcode oligonucleotide as a primer.

A plurality of microfluidic compartments, wherein at least 1% of said compartments form a subset in which each compartment is a droplet comprising:

• (i) a first cell,
• (ii) one second cell or one cell-free expression system, the second cell or the cell-free expression system expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on a surface of the first cell, and
• (iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and/or of the one second cell or the one cell-free expression system,
• wherein the compartment volume of the subset and the cell concentration of the first cell (i), the cell concentration or cell-free expression system concentration of the one second cell or the one cell-free expression system (ii), and the set concentration of the set of barcode oligonucleotides (iii) is such that λ is between 0.1 and 2 for each of the cell concentration of the first cell (i), the cell concentration of the one second cell or cell-free expression system concentration of the one cell-free expression system (ii), and the set concentration of the set of barcode oligonucleotides (iii),
• wherein λ is the average number of cells, cell-free expressions systems and sets of the barcode oligonucleotides per compartment volume of the subset.