GSM9450974: Pig#25,NRP+SCS 2h; Sus scrofa; RNA-Seq
Total RNA was extracted using the Rneasy Mini Kit (Qiagen) as per manufacturer guidelines. RNA purity was assessed on a NanoDrop spectrophotometer. RNA samples were quantified using Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA inputs were normalized to 100ng. ERCC RNA Spike-In Mix (Cat: #4456740) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer's protocol. RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads.  Enriched mRNAs were fragmented for 15 minutes at 94 °C. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq instrument according to manufacturer's instructions.  The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software.