Whole Genome Shotgun Sequencing of Sus scrofa: blood serum
RNA for NGS analysis was isolated from 200 l serum using the MagMAX CORE Nucleic Acid Purification Kit and a KingFisher Flex device according to the manufacturers instructions (Thermo Fisher Scientific, Waltham, USA). Each sample was prepared twice, with and without a DNase treatment. For the DNase digestion the script of the manufacturer was extended. After the second washing step the beads were incubated for 15 min at RT with the DNase solution (10 l DNase Stock I + 70 l RDD; Qiagen RNase free DNase Set; Qiagen, Hilden, Germany). After two further washing steps the beads were eluted at 65C in 90 l elution buffer. The whole genome sequencing of the serum pool samples 7 and 8 was performed on demand using NGS technology (CeGaT, Tbingen, Germany). Both samples were chosen based on the results of the previous Sanger sequencing. As quality controls, the RNA content, concentration and RIN were determined using Qubit RNA (Thermo Fisher Scientific, Waltham, USA) and the Bioanalyzer RNA Pico (Agilent, Santa Clara, USA), respectively. For library preparation with the NuGen Trio RNA-Seq Library Preparation Kit (Tecan Genomics, Redwood City, USA), an amount of 0.82.5 ng RNA was used. Sequencing was carried out by the MiSeq 300 cycle v2 Sequencing Kit (Illumina, San Diego, USA) with 2 x 150 bp, yielding QC-values of 89.56% Q30 (Supplementary Table S1).