*4.9. RNA Extraction and Real-Time PCR*

For real-time PCR analysis, mRNA was isolated from day 16 sheep TE, OTR cells, and iOTR cells using RNeasy Mini Kit (Qiagen Inc. Germantown, MD, USA), following the manufacturer's protocol. The mRNA was reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time PCR reactions were run in triplicate in 384-well plates, using 10 µL reaction volume in each well. The reaction volume included 5 µL of 2x Light-Cycler 480 SYBR Green I Master (Roche Applied Science, Penzberg, Germany), 50 ng reverse-transcribed mRNA, and 1 µM of target-specific forward and reverse primers. Primer sequences used for real-time PCR are listed in Supplementary Table S2. PCR reactions were incubated in the Light-Cycler 480 PCR machine (Roche Applied Science, Penzberg, Germany) at the following cycling conditions: 95 ◦C for 10 min, 45 cycles of 95 ◦C for 30 s, 55 ◦C for 1 min, and 72 ◦C for 1 min. Relative mRNA levels were normalized using *RPS15.* For miRNA profiling, total RNA was extracted using a miRNeasy Mini Kit (Qiagen Inc. Germantown, MD, USA), following the manufacturer's protocol. Then, 300 ng total RNA was reverse-transcribed to cDNA using miScript RT II kit (Qiagen Inc. Germantown, MD, USA). Real-time PCR reactions were run in triplicate in 384-well plates, using 10 µL reaction volume in each well. The reaction volume included 5 µL of 2x QuantiTech SYBR Green Master Mix (Qiagen Inc. Germantown, MD, USA), 3 ng cDNA, 1x miScript universal primer (Qiagen Inc. Germantown, MD, USA), and 1x miScript assay for *let-7* miRNAs (*let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i*). These reactions were incubated in the Light-Cycler 480 PCR machine (Roche Applied Science, Penzberg, Germany) at the following cycling conditions: 95 ◦C for 15 min, 45 cycles of 94 ◦C for 15 s, 55 ◦C for 30 s, and 70 ◦C for 30 s. Relative miRNA levels were normalized using *SNORD-48*.
