CPC C12N 15/1065 (2013.01) [C12N 15/115 (2013.01); C12Q 1/6806 (2013.01); C12N 2310/3517 (2013.01)] | 11 Claims |
1. A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells or cell nuclei, comprising:
a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
b. contacting the one or more nucleic acids with a first nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of first extension products comprising the oligonucleotide-tethered dideoxynucleotide at the 3′ end;
c. annealing a splint oligonucleotide which is at least partially complementary to the tethered oligonucleotide of the first extension products, and
d. contacting the first extension products with a nucleic acid polymerase and one or more nucleotides to allow the polymerase to extend across the annealed splint from the 3′ hydroxyl of the tethered oligonucleotide to produce a second extension product, thereby producing a library of nucleic acids,
wherein if the sample comprises a plurality of cells or cell nuclei, the cells or cell nuclei are fixed and permeabilized prior to annealing the first primer.
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