RNA isolation and detection were carried out as previously described (Mayerle and Guthrie 2016 (link)). Briefly, cultures were grown at 30°C overnight to saturation and then diluted to an OD600 of approximately 0.1. Diluted cultures were allowed to grow to an approximate OD600 of 0.5, pelleted, washed briefly with water, and then snap frozen at −80°C for further processing. Total cellular RNA was isolated using hot acid phenol followed by ethanol precipitation, and 20 µg total RNA was treated with 15 µL RNase free DNase I (NEB) in a total volume of 250 µL for 1 h at 37°C, and then re-extracted with phenol–chloroform. Superscript III Reverse Transcription System (Invitrogen) dN9 primers (Life Technologies) were used in all reverse transcription reactions. qPCR was performed using NEB Taq Polymerase and the gene-specific primers listed in Table 3, or previously described NSP1 and SEC17 primers. qPCR cycle conditions were 95°C for 3 min, 39 cycles of 95°C for 15 sec, 55°C for 30 sec, 72°C for 15 sec, followed by one cycle at 72°C for 3 min. Three biological replicates using total RNA isolated from separate biological cultures were assayed in technical triplicate.