GSM2160051: Bos_taurus_15 (30M); Bos taurus; RNA-Seq
longissimus muscle from each individual were harvested for RNA isolation within 10 minutes after slaughter. All fresh tissue samples were collected and divide it into 1.5 mL plastic centrifuge tube (each sample weighing around 100 mg ) and snap frozen in liquid nitrogen for RNA-seq. two cDNA libraries were constructed from pooled Emb135 (n=3) and 30M (n=3) samples.Messenger RNA was isolated from the total RNA samples using oligo (dT) magnetic beads (Invitrogen). The purified mRNA was first fragmented to approximately 200-700 nt fragments using the RNA fragmentation kit (Ambion). First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase. After the first strand was synthesized, a custom second-strand primer and strand synthesis buffer (Illumina) were added, followed by dNTPs, RNase H and DNA polymerase I to nick translate the second-strand. The reaction was then purified using a QiaQuick PCR column (Qiagen) and eluted in EB buffer (Qiagen); the samples were then subjected to end repair, addition of a single A base, adaptor ligation, agarose gel isolation of approximately 200-bp cDNA, and PCR amplification. Finally, the samples were sequenced using the Illumina HiSeq™ 2000 platform.