title: RNA seq of sus scrofa: longissimus dorsi
design: Three micrograms of total RNA was used to isolate poly-A RNA using the Dynabeads mRNA Direct Kit . The RNA fragmentation, library prep and adapter ligation were performed using the NEBNext Ultra Directional Library Prep kit for for Illumina (New England Biolabs, Ipswich, MA). Library size and quality were assessed using the High Sensitivity DNA Analysis Kit for the Agilent Bioanalyzer (Agilent, Santa Clara, CA) and library concentration was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Single-read 75-bp sequencing of libraries were performed using a NextSeq500 (Illumina, San Diego, CA). The FastQC app in BaseSpace was used to assess read quality and the FASTQ Toolkit was used for adapter trimming, base trimming low quality reads at the 3 end, and filtering using a minimum mean quality score of 30 (Illumina) (http://basespace.illumina.com/apps/). Alignment was performed using the Star Alignment app in BaseSpace and aligned to the pig genome (sus scrofa 11.1_v91) (Table S1 and Figure S2). The resulting Bam files were uploaded into SeqMonk (v.1.37.1, http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/.)