Taq mastermix

Total RNAs from porcine cells and tissues were extracted by Trizol Reagent (Invitrogen). RNAs were examined by measuring OD260/280 ratio, and samples with a ratio around 2.0 were used for reverse transcription. Two microgram total RNAs were reverse-transcribed with oligo-dT primer (Thermo Fisher) using RevertAid^TM reverse transcriptase (Thermo Fisher). RT-PCRs were performed using 2 × Es Taq MasterMix, 2 × Taq MasterMix (CW Biotech, China) for 32 cycles at 94 °C 30 s, 57 °C 30 s, and 72 °C 45 s. The negative control was done by directly performing PCR with total RNAs to check the contamination of genome DNA. Quantitative RT-PCRs (qRT-PCR) were performed using a 10-fold dilution of cDNA with SYBR Green PCR Master Mix (Takara Biotechnology, China), and detected with StepOnePlus Real-Time PCR System (Applied Biosystems). Reactions were performed for 40 cycles at 95 °C 15 s and 60 °C 60 s, and measurements were performed on three biological replicates and each reaction was performed in triplicate. The expression level of target gene was normalized to the expression level of beta-actin. Melting curve analysis was conducted to confirm the specificity. Primers used in this study are listed in Supplementary Table S1.

A total of 5×10⁶ cells at 75% confluence were harvested for genomic DNA isolation with the Tissue DNA Kit (OMEGA, D3396-02, USA) following the manufacturer’s instructions. The extracted DNA was amplified by PCR in a total volume of 25 µl containing 2X Taq Master Mix (CW Biotech, Beijing, China) 12.5 µl, forward primer 1 µl, reverse primer 1 µl, DNA 500 ng and RNase free water. The primers used are shown in Table 1. The conditions for PCR were as follows: 98°C for 3 mins; 98°C for 10 s, 60°C for 20 s and 72°C for 20 s for 35 cycles; 72°C for 10 mins; and a 4°C hold. The PCR products were cloned into the pMD19-T vector (D102A, TaKaRa, China) and sent to BGI (Shenzhen, Guangdong, China) for sequencing.

R gene-specific primer sets were designed based on their transcriptome sequences to perform PCR amplification using gDNA from TA7733 and CS as templates to verify 2M^b specific genes. PCR amplification were conducted in 15 μl reaction volumes containing 2 μl template gDNA (100 ng/μl), 0.25 μl forward primer (10 μmol/l), 0.25 μl reverse primer (10 μmol/l), 7.5 μl Taq MasterMix (CW Bio Inc., China) and 5 μl ddH₂O. PCR cycling conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 50–66°C for 30 s, and 72°C for 1 min, followed by a final 10-min extension at 72°C. The PCR products were digested with four base-restriction enzymes. Five microliters of a restriction enzyme mixture containing 2.8 μl of ddH₂O, 2.0 μl of CutSmart buffer, and 0.2 μl of an enzyme stock solution was added to 15 μl of PCR products and incubated for 3.5 h at 65°C. The PCR or restricted PCR products were separated on a 2.0% agarose gel-electrophoresis stained with ethidium bromide and visualized by UV light.