qPCR and HRM Assay Protocol

qPCR was performed in a Bio-Rad CFX96 using the primers in S6 Table with the Dynamo HS SYBR Green qPCR Kit (ThermoFisher, Waltham, MA USA). Templates were either genomic DNA or cDNA prepared using the Maxima First-Strand cDNA Synthesis kit (ThermoFisher) with RNA treated with RQ1 DNAse (Promega, Fitchberg, WI USA). Reactions were performed in triplicate in a volume of 20 μl using 10 ng of template, 0.2 μM of each primer, and 40 cycles of 10 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. For RT-qPCR, constitutive gene 09862 (GenBank accession NW_003303742; [102 (link)]) was used for normalization, and parallel reactions were performed using RNA without reverse transcriptase treatment as a negative control. HRM assays were performed using the same cycling conditions with the primers shown in S6 Table, but using the Bio-Rad Precision Melt Supermix kit. Melt curves were generated using Bio-Rad Precision Melt Analysis software.