Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Total RNA was extracted from samples of biceps femoris muscle using TRIzol Reagent (Invitrogen). The concentration and quality of RNA was confirmed using an Agilent 2100 system and agarose gel electrophoresis (AGE). Equal amounts of RNA from the tissue samples of three abnormal cloned piglets, three normal cloned piglets were mixed into three pools. The two independent RNA pools were then used for library construction and sequencing by the Beijing Genome Institute. Briefly, after evaluation of the quality of total RNA, mRNA was enriched by using oligo(dT) magnetic beads. After adding the fragmentation buffer, mRNA was interrupted to short fragments (˜200 bp). First-strand cDNA was synthesized using a random hexamer primer using the mRNA fragments as templates. Then buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. Double-strand cDNA was purified with a QiaQuick PCR Extraction kit and washed with Ethidium Bromide buffer for end repair and addition of single nucleotide A (adenine). Finally, sequencing adaptors were ligated to the fragments. Required fragments were purified by AGE and enriched by PCR amplification.