Syto9 dye

Genomic DNA and DNA from FA-SAT clones, both of high purity, were analysed by High-Resolution Melting Assays (HRM) with MeltDoctor HRM Master Mix, which uses the SYTO9 dye (Applied Biosystems, Thermo Fisher Scientific), and with the 83-bp B and 83-bp C primers (supplementary table S1, Supplementary Material online). All the PCR mixtures were done according to the recommendations of the manufacturer. Experiments were performed in the StepOne real-time PCR system (Applied Biosystems, Thermo Fisher Scientific) with the next program: initial denaturation at 95 °C (10 min), 40 cycles of 95 °C for 15 s, followed by 59 °C for 1 min. Subsequently, a melt curve was performed with a denaturation step of 95 °C for 15 s, an annealing of 59 °C for 1 min and a high-resolution melting of 95 °C for 15 s with a continuous ramp rate of 0.3%, followed by a final step of annealing of 60 °C for 15 s. All reactions were performed in triplicate, and negative controls (without template) were also included on the plate. The StepOne software (version 2.2.2, Applied Biosystems, Thermo Fisher Scientific) was used for data acquisition and the High Resolution Melt software (version 3.0.1, Applied Biosystems, Thermo Fisher Scientific) for data analysis (we used the standard software parameters suggested by the manufacturer).

All HRM-qPCR assays were carried out in a RotorGene Q machine (Qiagen, Hilden, Germany) in a 10 μl reaction volume consisting of 1 μl of a specific concentration of DNA template, 0.2 μM of each primer, 5 μl MeltDoctor™ HRM master mix containing SYTO 9 dye (Applied Biosystems, California, USA) and 3.6 μl sterile, deionized water. The thermal profile for each qPCR consisted of a pre-PCR step of 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 15 s and 30 s at the annealing temperature of 60 °C. Immediately following qPCR, HRM was carried out to identify the species using the melt curve analysis program. The HRM conditions were as follows: an increase in temperature from 60 $°$ C to 95 °C by 0.2 °C increments with a 2-s hold after each step. The average melting temperature (T_m) for each parasite species was determined using the High-Resolution Melt Software v 3.01 (Applied Biosystems, California, USA).

qPCR experiments were performed in Corbett RotorGene 6000 Real-Time PCR instrument (Corbett Research) using SYTO 9 dye (Invitrogen). The sequences of the primers are included in Supplemental Table 1. Random decamer primed cDNA was used for the qPCR experiments involving host genes, hypoxia responsive genes, and Drosha mRNA quantitation. miRNA quantitation was performed using ABI TAQMAN miRNA quantitation kit (Applied Biosystems) according to the manufacturer's protocol. For the identification and quantitation of alternatively spliced transcripts, RT-PCR was performed using oligo-dT primed cDNA. Appropriate minus RT controls were included in every experiment to ensure absence of genomic DNA contamination.