RNA was DNase treated using ezDNase™ enzyme (Thermo Fisher) following the published protocol. cDNA was immediately synthesized using the iScript™ Select cDNA Synthesis Kit (Bio-Rad) with random primers following the published protocol and stored in TE buffer. Pluripotency markers were analyzed at a concentration of 0.3 ng cDNA, while germ layer and cardiac markers were analyzed at 3 ng. All polymerase chain reactions (PCRs) were carried out using the SsoAdvanced™ Universal SYBR® Green Supermix Kit (Bio-Rad) according to the published protocol with an annealing temperature of 60°C for 15 s and primers specifically designed for rhinoceros (Supplementary Table S3). Primer concentration was checked for all primers and optimized for ACTB to be 250 nM forward and 300 nM reverse, while all the others are used at 200 nM. To eliminate any nonspecific signal from primer-dimer formation, an additional 80°C read step for 5 s was added after annealing. By reading the signal at 80°C instead of 60°C, all smaller amplicons would have been denatured and would not contribute to expression levels. The temperature was chosen based on the melt curves of the GOI and was 1.5°C below the lowest observed GOI melt temperature. Primer specificity and efficiency were checked with a standard curve of mixed cell types and melt curve analysis. Each sample was run in triplicate, and the expression levels were normalized to ACTB (actin beta), which is an accepted housekeeping gene in horse [23,31] and human [32] iPSC analyses. Samples that did not amplify strongly enough to produce a melt curve were subsequently removed from the analysis.

Loci in which genomic amplification was observed in the no reverse transcriptase (NRT) reaction with a Cq value <5 cycles from the sample were corrected using the following formula [33]:

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