454 sequencing of EF library
cDNA was synthesized using the SMART cDNA library construction kit (Clontech, CA, USA). First-strand cDNA was synthesized for each library from 0.5-1.0 µg of total RNA in a 10-*l reaction as described in the kit protocol using the SMART IV primer (AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG), a modified oligo(dT) primer (TAGAGACCGAGGCGGCCGACATGTTTTGTTTTTTTTT CTTTTTTTTTTVN), where V = A, G, or C and N = A, G, C, or T), and SuperScript II reverse transcriptase (Invitrogen, CA, USA). Double-stranded cDNA was synthesized using the modified oligo(dT) primer and the SMART 5* PCR primer (AAGCAGTGGTATCAA CGCAGAGT) followed by a SfiI digestion as described in the SMART kit protocol. Amplified cDNA was purified using the QIAquick purification kit (Qiagen, CA, USA). All column elutions for a specific library were pooled, and the relative cDNA concentration was estimated running a 1% agarose gel electrophoresis with ethidium bromide staining and comparison to a standard molecular weight ladder.