Figure 2.

A scheme of cDNA library construction based on RC. Steps involved in RC-based cDNA library construction are schematically shown and were almost identical to those described in the instructions of SuperScript Plasmid System (Life Technologies) except for the use of the attB2-dT adapter primer, attB1 adapter and inclusion of an in vitro recombination reaction. Oligonucleotide sequences used for the attB2-dT primer adapter, upper and lower strands of attB1 adapter are as follows: attB2-dT primer, 5′-GCGAAGCCCACCACTTTGTACAAGAAAGCTGGGCGGCCGC(T)₂₀-3′; upper and lower strands of attB1 adapter, 5′-TCGACGCGTACAAGTTTGTACAAAAAAGCAGGCTCTTC-3′ and 5′-GAAGAGCCTGCTTTTTTGTACAAACTTGTACGCG-3′, where the attB1 and attB2 sequences are underlined. An expected reaction product using phosphorylated attB1 adapter is illustrated. In the phosphorylated attB1 adapter, the 5′-end of only the lower strand of the attB1 adapter was chemically phosphorylated.