5′-RLM-RACE for TSS identification

For RACE, RNA was isolated from BoHV-1 infected MDBK cells in 25 cm² cell culture flask at two hours post infection. RACE was performed using 5′-RLM-RACE (RNA Ligase Mediated Rapid Amplification of cDNA Ends) kit (Ambion, Life Technologies) according to the manufacturer’s recommendations. In brief, 5 µL of cDNA was subjected to the first round of PCR amplification using the adaptor primer (outer) provided in the kit and gene-specific reverse primer. The reaction was carried out in a thermal cycler (Biometra, U.K.). The cycling conditions were denaturation at 95 °C for 2 minutes, followed by 34 cycles of denaturation at 95 °C for 30 seconds, annealing touchdown (from 65 to 55 °C) for 45 seconds and extension at 72 °C for 1 minute, with a final extension at 72 °C for 5 minutes. After amplification, the reaction products were diluted 20-fold with water, and 3 µl DNA were then re-amplified for 30 cycles using the same reaction conditions with nested and adaptor primer (inner primer provided in the kit). Positive PCR products were eluted from 1% agarose gel and ligated into pGEM-TEasy vector (Promega) and sequenced through vendors (Bioserves).