Single cell analyses

A method for producing a library of nucleic acids containing universal primer sites on the 5′ and 3′ end from input nucleic acids comprising:

• (a) providing a picowell array comprising a plurality of picowells,
• (b) contacting input nucleic acids with a pool of capture oligonucleotides in a picowell of the plurality of picowells, each capture oligonucleotide in the picowell containing a 5′ universal primer site and a 3′ target binding site complementary to a nucleotide sequence in an input nucleic acid, wherein the input nucleic acids comprise RNA, and wherein the 3′ target binding site contained in the capture oligonucleotide comprises a poly(dT) sequence,
• (c) adding a DNA polymerase and thereby extending the capture oligonucleotides hybridized to the input nucleic acids, to form first strand cDNA nucleic acids each comprising the 5′ universal primer site and a sequence that is complementary to one of the input nucleic acids,
• (d) contacting the first strand cDNA nucleic acids with a pool of second strand priming oligonucleotides, each consisting of a 5′ universal primer site and a 3′ target binding site complementary to a nucleotide sequence in the first strand cDNA nucleic acid, wherein each 3′ target binding site of the second strand priming oligonucleotides consists of a random sequence,
• (e) adding a DNA polymerase and thereby extending the second strand priming oligonucleotides, to form second strand cDNA nucleic acids comprising 5′ and 3′ universal primer sites that flank nucleotide sequences present in the input nucleic acids, and
• (f) amplifying the second strand cDNA nucleic acids comprising 5′ and 3′ universal primer sites formed in step (e).