Qiazol protocol

Total RNA was extracted from frozen liver samples following the QIAzol protocol (Qiagen, Germany) and further cleaned with the RNeasy MinElute cleanup kit (Qiagen, Germany). Quality and quantity of total RNA were determined using the 2200 TapeStation from Agilent (USA). Each RNA sample used had a RINe (RNA integrity) value above 7. RNA was extracted from 5 individual liver samples (n = 5) per ploidy group at selected sampling stages fry (1455 degree-days post-start feeding, ddPSF), parr (1888 ddPSF) and smolt (2745 ddPSF). RNA-seq libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA Library Prep kit (NEB, USA). The barcoded libraries were checked for quality and quantity on a 2200 TapeStation before pooling and sequencing. Library pools were sequenced on the NextSeq500 (Illumina, USA) with the Nextseq 500/550 high output kit v2.5 sequencing kit (150 cycles single-end reads) at Nord University, Norway.

RNA was extracted using RNeasy Plus Mini kit or QIAzol protocol (Qiagen, Milan, Italy) and converted to cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Monza, Italy). qRT-PCR was performed using the 7900 HT Fast Real Time PCR system (SDS version 2.3 software) or CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Segrate, Italy) using commercially available primers (TaqMan Gene Expression Assays; Thermo Fisher Scientific, Monza, Italy) listed in Text S1. Relative gene expression was calculated as described [46 (link)].

Ten million Molt-3 cells continuously infected with HHV-6B were harvested. Total RNA was isolated using the standard Qiazol protocol (Qiagen Inc.) and processed for mRNA analysis by reverse transcriptase PCR (RT-PCR). In brief, 1 μg of total RNA was reverse transcribed in a total volume of 20 μl using the Moloney murine leukemia virus reverse transcriptase. One-tenth of the cDNA was used for the amplification of specific splicing junctions, using the primers specified in Table 5. PCR products were amplified, using Q5 high-fidelity DNA polymerase (New England BioLabs Ltd., Whitby, ON, Canada), at 98°C for 1 min, 40 cycles of 10 s at 98°C, 30 s at 60°C, and 3 min at 72°C, and a final elongation step at 72°C for 2 min. PCR products were then run on a 2% agarose gel and visualized using a Gel Doc XR+ System (Bio-Rad Laboratories Canada Inc., Mississauga, ON, Canada).