title: RNA-Seq of Ovis aries: abomasum organoids + IL-13
design: Ovine abomasum organoids were exposed to four different conditions for 48 h before RNA extraction and sequencing i) Abomasum organoids (CONTROL) ii) Stimulated with IL-13 (IL13) iii) Stimulated with IL-13 and transfected with miRNA-mimic-control (CONTROL MIMIC) iv) Stimulated with IL-13 and transfected with miR-5352-mimic (MIMIC). Four biological replicates each were used (n=4). Total RNA was extracted using RNeasy Micro Kit (QIAGEN) from three wells of ~ 300 organoids, following manufacturer's instructions. The RNA concentration was determined using Qubit RNA High Sensitivity (HS) (Thermo Fisher), and quality and integrity determined using Bioanalyzer. For each sample, 1 g of total RNA was used for RNA-seq analysis. All library synthesis and sequencing were conducted at BGI (Hong-Kong). In brief, oligo dT beads were used to enrich for poly A mRNA, first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP. The synthesized cDNA was subjected to end-repair and was then 3' adenylated and adaptors ligated to the ends of these 3' adenylated cDNA fragments. For PCR amplification, dUTP-marked strand was selectively degraded by Uracil-DNA-Glycosylase (UDG) and the remaining strand was amplified to generate a cDNA library suitable for sequencing. The PCR product was desaturated by heat and the single strand DNA was cyclized by splint oligo and DNA ligase. The DNBseq technology was used with a sequencing length of PE 150.