Mastercycler nexus gx2 thermal cycler

Total RNA was extracted from cultured splenocytes using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and quantified using the QIAxpert system (Qiagen). Complementary DNA was synthesised by reverse transcribing total RNA (400 ng) using the GoScript™ Reverse Transcription system (Promega, Madison, WI, USA) in a MasterCycler^® Nexus GX2 thermal cycler (Eppendorf). The primer sequences were as follows: minipig GAPDH, 5′-GGGCATGAACCATGAGAAGT-3′ and 5′-TGTGGTCATGAGTCCTTCCA-3′; IL-4, 5′-AGAACACGACGGAGAAGGAA-3′ and 5′-TTGCCATGCTGCTAGGTT-3′; IFN-γ, 5′-CAGCTTTGCGTGACTTTGTG-3′ and 5′-TTTTGTCACTCTCCTTCCAAT-3′; and TNF-α, 5′-CCCCCAGAAGGAAGAGTTTC-3′ and 5′-CGGGCTTATCTGAGGTTTGA-3′. The qRT-PCR was performed on a QuantStudio™ 5 Real-Time system (Thermo Scientific, Wilmington, DE, USA) with Power SYBR Green Master Mix (Thermo Scientific). The transcript level for each gene was normalised to that of GAPDH.

Total genomic DNA from fins was extracted by proteinase K digestion, followed by a simplified DNA isolation procedure^{66 (link)}. DNA quality and quantity were assessed by spectrophotometry (IMPLEN N50, Germany), and each sample was diluted to 15 ng μL⁻¹ in DNAse/RNAse free water. Total data set containing 1586 individuals from 20 locations was characterized using two multiplex PCRs, SMsa-1 and SMsa2 (SuperMultiplex Sparus aurata), developed by Lee-Montero et al.^{30 (link)} with 21 specific microsatellite markers applied (Supplementary Table S1). Using the Multiplex PCR kit (Qiagen, Germany), amplification of the loci was run in 10 μL reactions containing 15 ng DNA on an Eppendorf Mastercycler Nexus Gx2 thermal cycler, having final concentrations of all primers uniformly set at 0.2 μM. For both multiplex PCRs, conditions were as follows: initial denaturation at 95 °C for 5 min, 26 cycles at 95 °C for 30 s, annealing at 60 °C for 90 s, and elongation at 72 °C for 30 s; and then a final elongation of 30 min at 60 °C. Fragments were separated on an ABI3130 automated sequencer (Applied Biosystems) while electropherograms and genotypes were analysed using GeneMapper software v.3.5 (Applied Biosystems).