In Vitro Transcription and Microinjection of Fluorescent Reporter RNAs

Three RNAs encoding LUC and EGFP were synthesized and purified as previously described^{43 (link)}. Briefly, pCS2 plasmids, pCS2-LUC-EGFP-pA, pCS2-nt5ea-LUC-EGFP-pA, and pCS2-nt5eb-LUC-EGFP-pA (Fig. S1a,b), were linearized with NotI and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) for in vitro transcription. The capped RNA was transcribed from the purified DNA templates using the Message mMachine SP6 Kit (Life Technologies, Carlsbad, CA, USA). The synthesized RNA was purified using an RNeasy Mini Kit (Qiagen) for microinjection. Approximately 2–4 nL of 100 ng/µL of each RNA was injected into the cytosol of embryos at the one-cell stage, as previously described^{44 (link)}. The injected embryos were observed under an SZX16 fluorescence stereomicroscope with the SZX2-FGFP filter set (Olympus, Tokyo, Japan). Microscopic images were captured using a DP73 camera and cellSens image acquisition software (Olympus).