Recombinant Fusion Proteins and Libraries from Immune Cell Repertoires
Disclosed herein are methods and compositions for generating a repertoire of recombinant fusion polypeptides from immune cells, and uses thereof.
1 . A composition comprising a pool of at least 10,000 monodisperse aqueous droplets in an oil solution,
wherein said aqueous droplets each have a diameter of between 1 micron and 200 microns, wherein each of said aqueous droplets have an outer boundary comprising a surfactant, and
wherein a plurality of said at least 10,000 monodisperse aqueous droplets comprise
a first probe comprising a first polynucleotide having a length of between 15 and 120 nucleotides, wherein said first polynucleotide is complementary to a first subsequence of a polynucleotide encoding a constant domain or poly(A) tail of an antibody or T cell receptor, and
a second probe comprising a second polynucleotide having a length of between 15 and 120 nucleotides, wherein said second polynucleotide is complementary to a second subsequence of a polynucleotide encoding a constant domain or poly(A) tail of an antibody or T cell receptor;
wherein said first and second probes are attached to at least one particle.
2 . The composition of claim 1 , wherein said first and second nucleic acid probes are bound to a particle.
3 . The composition of claim 2 , wherein said particle is a bead comprising agarose, glass, chemical polymers, or magnetic materials.
4 . The composition of claim 2 , wherein said probes comprise biotin and wherein said particle comprises streptavidin bound to the surface of the particle.
5 . The composition of claim 2 , wherein said composition comprises a covalent bond between said first probe or said second probe and said particle.
6 . The composition of claim 1 , wherein each of said plurality of monodisperse aqueous droplets further comprise reagents for overlap extension RT-PCR.
7 . The composition of claim 1 , wherein said first probe is bound to a polynucleotide encoding a light chain variable domain from a single isolated cell, and wherein said second probe is bound to a polynucleotide encoding a heavy chain variable domain from said single isolated cell.