GSM1423154: E3069; Ovis aries; RNA-Seq
Frozen conceptus samples were homogenized in RLT plus buffer and RNA was isolated using RNeasy Plus kit (Qiagen). RNA samples were depleted of ribosomal RNA using Ribominus system (Ambion). 20ng of ribosomal depleted sample and the Ion Total RNA-seq Kit v2 (Life Technologies) were used to construct strand specific sequencing libraries, with several deviations from the protocol.  Enzymatic RNA fragmentation was carried out at 37 C for 90 seconds.  Fragmented RNA was purified using 1.0X AMPure XP beads (Beckmann-Coulter Genomics).  cDNA and the final libraries were purified with 0.8X AMPure XP beads.   Emulsion PCR was performed on an Ion One Touch 2 instrument, using the Ion P1 Template OT2 200 v3 reagents.  Sequencing beads were quantified and evaluated by flow cytometry (Millipore Guava Easy Cyte) with Sybr Gold (Molecular Bioprobes) before loading on an Ion P1 semiconductor sequencing chip.  Sequencing was performed on an Ion Proton using Ion P1 200 v3 sequencing reagents and 440 flows.  The barcoded libraries were pooled three to a chip for sequencing.  Base calling and primary analysis was performed using Torrent Suite 4.0.2