Single embryo RT-qPCR

Pig qPCR primers (PUMA, p21, NOXA, PERP, spCas9, and glyceraldehyde-3-phosphate dehydrogenase) were mixed until the final concentration of 0.1 μM. A single blastocyst was placed in PBS buffer, and the embryo was pipetted with 0.5 μL PBS into reverse-transcribed reaction buffer, amplified by the Single-Cell Sequence Specific Amplification Kit (Vazyme, P621). Following the manufacturer’s instructions, fluorescence qPCR was performed with AceQ Universal SYBR qPCR Master Mix (Vazyme, Q511-02). Table S2 shows the primer sequences.