title: GSM1551999: 1024626; Ovis aries; RNA-Seq
library_construction_protocol: Abomasal lymph node (ALN) tissue, recovered at slaughter, was immediately cut into pieces approximately 0.5 cm3 and submerged in 10 volumes of RNAlater® (Ambion). This was stored overnight at 4 °C followed by long-term storage at -80 °C. Total RNA was extracted from the ALN tissue using Sigma TRI Reagent® (Sigma Aldrich, UK) according to the manufacturer’s instructions. Small RNAs (<200 nucleotides) and residual genomic DNA were removed with the RNeasy Mini Kit (Qiagen, Germany) and an in-solution DNase digestion (RNase-free DNase set; Qiagen, Germany) according to the manufacturer’s instructions. RNA quality was assessed using an Agilent® RNA 6000 Nano Assay on the 2100 Bioanalyzer, and total RNA was quantified using the NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, UK). Illumina TruSeq™ libraries were prepared following the TruSeq™RNA sample preparation v2 guide (Part #15026495 Rev. B) for total RNA with the following modifications: (i) the number of PCR cycles was reduced to 10 to minimise overcycling and (2) to avoid bead contamination the PCR products were purified using a Qiagen MinElute column rather than AMPure XP beads. Libraries were visualised using an Agilent® DNA 1000 assay on the 2100 Bioanalyzer, and quantified using the Qubit® dsDNA BR assay (Invitrogen, UK) according to the manufacturer’s instructions. The indexed cDNA libraries containing the specific Illumina TruSeq adapters were sent to GATC Biotech (Kontanz, Germany), where they were pooled. Each pool was sequenced on two lanes of an Illumina HiSeq2000 with 50 bp paired-end reads.