Superscript riii first strand

Total RNA was isolated from HH10 chick embryos using a Trizol kit (Invitrogen, USA) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized to a final volume of 25 μl using SuperScript RIII first-strand (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed as described previously^{29 (link),30 (link)}. The sets of primers used for RT-PCR are described in the Supplementary Fig. 1. The PCR reactions were performed in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The final reaction volume was 50 μl, comprising 1 μl of first-strand cDNA, 25 μM forward primer, 25 μM reverse primer, 10 μl PrimeSTARTM Buffer (Mg2+ plus), 4 μl dNTP Mixture (TaKaRa, Japan), 0.5 μl PrimeSTARTM HS DNA Polymerase (2.5 U/μl TaKaRa, Japan) and RNase-free water. cDNA was amplified for 30 cycles. One round of amplification was performed at 94 °C for 30 s and then 30 s at 58 °C and 30 s at 72 °C. The PCR products (20 μl) were resolved using 1% agarose gels (Biowest, Spain) in 1× TAE buffer (0.04 M Trisacetate and 0.001 M EDTA) and 10,000× GeneGreen Nucleic Acid Dye (TIANGEN, China) solution. The resolved products were visualized using a transilluminator (SYNGENE, UK), and photographs captured using a computer-assisted gel documentation system (SYNGENE). Each of these experiments was replicated at least three times.