Porcine Transcriptome Profiling by RNA-Seq

RNA libraries, with equal RNA amounts, from five curly-coated Mangalitza, seven Mangalitza×Angeln Saddleback shoats, and seven straight-coated individuals (see Supplementary Table S1) were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, United States) and indexed by NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs, NEB, Ipswich, United Kingdom). The prepared libraries were set in equimolar dilutions and submitted for sequencing for 66 million reads. In total, 32 libraries were sequenced on an Illumina NextSeq500 for 2 × 75 bp, further 15 libraries were sequenced on an Illumina NovaSeq6000 for 2 × 100 bp. In the next step, the binary base call (BCL) sequence output files were converted to FASTQ format and underwent quality control using FastQC version 0.11.8 (Andrews, 2010 ). FASTQ files were further processed using fastp version 0.20.0 (Chen et al., 2018 (link)) to control for adaptor content and discarding long stretches of N base >50 (--n_base_limit 50), 70% unqualified bases (unqualified_percent_limit 70), 1% complexity threshold (complexity_threshold 1) and low-quality reads (link)) based on Sus Scrofa11.1 genome reference (Ensembl, database version 101.111). In addition, STAR quantMode was run to extract raw gene counts.