| 主权项 |
1. A method for generating a directional cDNA library, the method comprising:
a) annealing one or more primers to a template RNA; b) extending the one or more primers in the presence of a reaction mixture comprising dATP, dCTP, dGTP, dTTP, and dUTP, wherein the reaction mixture comprises a ratio of dUTP to dTTP, wherein the ratio permits incorporation of dUTP at a desired density, thereby generating a one or more first strand complementary DNAs (cDNAs) comprising dUTP incorporated at a desired density; c) selectively cleaving the one or more first strand cDNAs comprising dUTPs incorporated at a desired density with uracil-N-glycosylase (UNG) and an agent capable of cleaving a phosphodiester backbone at an abasic site created by the UNG, wherein the cleaving generates a plurality of first strand cDNA fragments of a desired size comprising a blocked 3′ end; d) annealing a first adapter comprising a partial duplex and a 3′ overhang to a 3′ end of one or more of the plurality of first strand cDNA fragments comprising a blocked 3′ end, wherein the first adapter comprises a sequence A, and wherein the annealing comprises hybridizing a random sequence at the 3′ overhang to a complementary sequence present at the 3′ end of the one or more of the plurality of first strand cDNA fragments comprising a blocked 3′ end; e) extending the 3′ overhang hybridized to the complementary sequence with a DNA polymerase, wherein one or more double stranded cDNA fragments comprising the sequence A at one end is generated; f) ligating a second adapter comprising a sequence B to the one or more double stranded cDNA fragments comprising the sequence A at one end, wherein the ligating generates one or more double stranded cDNA fragments comprising the sequence A at one end and the sequence B at an opposite end, thereby generating the directional polynucleotide library; and g) optionally, amplifying and/or sequencing the directional cDNA library. |
