Poly (A) mRNA was depleted from P. marinus total RNA and a poly (A) tail was added to the remaining population using Escherichia coli Poly (A) Polymerase (Takara Mirus Bio) as reported [8] . First-strand cDNA synthesized using GeneRacer Oligo dT primer (Invitrogen) was used as PCR template. Two rounds of touch-down PCR were carried using the same conditions as above, with the extension time of 5 sec at 72uC. The first round of PCR was performed using PmaSL-LSF1 and GeneRacer3 primers. The PCR products were diluted 100-fold and used in the second round PCR with PmaSL-LSF2, PmaSL-LNF2, PmaSL-LNF3, PmaSL-S2F2, or PmaSL-S2F3, each paired with GeneRacer3, as the nested primers (Table 1) .