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The poly (A)+ RNA was dephosphorylated with bacterial alkaline phosphatase (BAP) and then decapped with tabacco acid pyrophosphatase (TAP). The decapped intact mRNA was ligated with DNA-RNA linker including SfiI site by treatment of T4 RNA ligase and the first strand cDNA was synthesized with Superscript II using SfiI oligo-dT primer. After first strand synthesis, RNA was degraded by NaOH treatment and cDNA was amplified by PCR reaction. The PCR products were digested with SfiI and cloned into DraIII- digested pME18S-FL3 vector. The obtained cDNA vectors were used for transformation of competent cells E. coli Top10F' by electroporation method. The cDNA libraries constructed by this method are full-length enriched cDNA library.
Nucleotide
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