Nanodrop 2000 spectrophotometer

Genomic DNA was isolated using a commercial extraction kit (Blood DNA isolation kit, Geneaid Biotech Ltd. New Taipei City, Taiwan) as recommended by the manufacturer. Quantity and quality of extracted DNA were checked using a NanoDrop 2000 spectrophotometer (VWR, Darmstadt, Germany). Polymerase chain reaction (PCR) for the amplification of a 970 bp fragment containing the complete PRNP coding region was done as described in Lühken et al. (2007 (link)) for ovine PRNP. Due to differences between sheep and goat PRNP sequences in the binding region of the forward primer, it was necessary to modify the forward primer (TGCCASTGCTATGCAGTCATT, changed nucleotides underlined). PCR program: An initial denaturing stage at 95 °C for 5 min was followed by 35 cycles at 94 °C for 30 s, 55 °C for 90 s, and 72 °C for 60 s, with a final step at 72 °C for 10 min. The PCR products were purified using a commercial kit (MSB Spin PCRapace, Stratec Molecular, Berlin, Germany), diluted to 10 ng/µl after quantification by agarose gel electrophoresis and sent to LGC Genomics GmbH (Berlin, Germany) for Sanger sequencing using the reverse PCR primer. The resulting chromatograms were analyzed with Chromas 1.74 (Technelysium Pty Ltd, South Brisbane, Australia). The GenBank sequence EF192309.2 was used as a reference sequence of the goat PRNP gene, representing the wild type (Acutis et al. 2008 (link)).

Total RNA was isolated using the GenElute^TM Mammalian Total RNA Miniprep Kit (ThermoFisher Scientific) including DNAse I treatment of samples to eliminate genomic DNA. Quality of RNA was determined by measuring OD₂₆₀/OD₂₈₀ by UV/Vis spectroscopy using a Nanodrop 2000 spectrophotometer (VWR International GmbH). RNA was transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Real-time PCR analysis was performed with ~10–30 ng of cDNA using TaqMan^® Universal PCR Master Mix and pre-designed TaqMan^® Gene Expression Assays (Supplemental Table 2). Reactions were carried out on a StepOnePlus^TM Real-Time PCR System (ThermoFisher Scientific). Cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and for 1 min at 60 °C. Data were analyzed according to the 2^−∆∆Ct method using cyclophilin D as reference gene. Lack of amplification was verified in no-template controls.

The RNA was first treated with Turbo DNAse (TURBO DNA‐free Kit, Life Technologies, AM1907) and then purified using DNAse Inactivation buffer. The RNA was then centrifuged for 1.5 min at 1,000 g and the supernatant was collected and centrifuged once more at the same condition. The RNA quantity was determined by measuring the absorbance at 260 nm (NanoDrop 2000 spectrophotometer; Peqlab).
Poly‐A selection was performed according to manufacturer’s instructions (Bio Scientific Corp., 710 NOVA‐512991). Following Poly‐A selection, mRNA libraries were prepared according to manufacturer’s instructions (Bio Scientific Corp., NOVA‐5138‐08), except that the RNA was incubated at 95°C for 13 min to generate optimal fragment sizes. The sequencing library quantity was determined using Qubit (Thermo Fisher Scientific). The library integrity was assessed with a Bioanalyzer 2100 system (RNA 6000 Pico kit, Agilent Technologies). The libraries on biological duplicates from each genotype were subjected to 75 base‐pair single‐end sequencing on Illumina NextSeq500 at the Center for Functional Genomics (CFG).