GSM2498618: T15A-RNA-Seq; Ovis aries; RNA-Seq
Total RNA was extracted from 30 mg of ground tissue by using hot phenol method. The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1DNase (Promega) to remove DNA. For each sample, 10μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Invitrogen) before used for directional RNA-seq library preparation. The purified mRNAs were then iron fragmented at 95°C followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified, amplified, and stored at -80°C until they were used for sequencing.