Universal primer a mix upm

The transcription initiation site (TSS) of the bovine ACSL1 gene was determined using a BD SMARTTM RACE cDNA amplification kit (Clontech Inc, CA, USA) according to the manufacturer’s instructions. Briefly, 1 μg of total RNA isolated from the longissimus thoracis was reverse-transcribed with PowerScript RT (Clontech Inc, CA, USA). PCR was performed using a Universal Primer A Mix (UPM) (Clontech Inc, CA, USA) and gene-specific primers (Supplementary Table S1) located in exons 2 and 3 of the ACSL1 gene. The primary PCR products were diluted 20-fold as the nested PCR template. The PCR products were separated by electrophoresis on 2% agarose gels containing 0.6 g/mL ethidium bromide and visualized under UV. The purified amplimers were cloned into T-Vector pMD19 (simple) (TaKaRa, Dalian, China) and 20 clones were sequenced.

The BD SMARTTM RACE cDNA amplification kit (Clontech Inc, Mountain View, CA, USA) was used to identify the bovine SLC27A1 transcriptional start site (TSS) based upon the manufacturer’s directions. In short, 1 μg longissimus thoracis RNA underwent PowerScript RT (Clontech Inc, Mountain View, CA, USA) reverse transcription, after which Universal Primer A Mix (UPM) (Clontech Inc, Mountain View, CA, USA) was added for PCR amplification along with appropriate primers (Table S1) that were specific for SLC27A1 exons 2, 3, and 4. The amplicons from this PCR reaction were subjected to a 20-fold dilution, followed by 2% agarose gel separation in a gel supplemented with 0.6 μg/mL ethidium bromide for UV visualization of the DNA. After purification, the amplified sequences were cloned into T-Vector pMD19 (simple) (TaKaRa, Dalian, China) and 20 clones were subject to sequencing.

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 described^{27 (link)}. For sequencing PCR products were separated by electrophoresis in 2% agarose gels and subsequently cloned into T-Vector pMD19 (simple) (Takara).