2.4. cDNA Synthesis and Amplicon Analysis

For the cDNA synthesis, 5 μL total RNA from each sample, together with an IBV gene-specific primer (5′-TACCGTTCGTTTCCA-3′), was subjected to reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s guidelines. Six individual amplicons were amplified using the following mixture: 10 μL LongAmp® Taq 2X Master Mix (New England Biolabs, Ipswich, MA, USA), 0.5 μL forward primer (10 pmol/µL), 0.5 μL reverse primer (10 pmol/µL), and 2 µL of cDNA in a final volume of 20 µL. The cycling conditions were as follows: 95 °C for 5 s, followed by 35 cycles of 95 °C for 45 s, 53 °C for 45 s, and 65 °C for 5 min, with a final extension at 65 °C for 10 min. The PCR products were run on a 1% agarose gel to confirm the presence of amplicons. The amplicons generated for each primer pair were pooled (in equal volumes) into a single mixture for library preparation and Illumina sequencing. The pooled mixture was purified/cleaned up using AMPure XP beads (Beckman Coulter, Brea, CA, USA) following the manufacturer’s instructions.