To identify the transcriptional start site (TSS) of the bovine SIX1 gene, 5’-RACE was performed on total RNA from the longissimus thoracis muscle using a BD SMARTTM RACE cDNA amplification kit (Clontech Inc., CA, USA) according to the manufacturer’s protocol. PCR was performed using a Universal Primer A Mix (UPM, Clontech Inc., CA, USA) and the nested PCR primers (Table S1 ) located in exon 1 of the SIX1 gene. The conditions and methods used were as previously described27 (link). For sequencing PCR products were separated by electrophoresis in 2% agarose gels and subsequently cloned into T-Vector pMD19 (simple) (Takara).
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