Message mmachine sp6kit

Ac, phiC31 integrase, and FLP expression vectors (Figure 1A) were linearized using NotI, purified using phenol-chloroform extraction, and used as template to synthesize capped mRNAs using the Message mMachine SP6Kit (Life Technologies, Gaithersburg, MD) as described previously (Ishikawa et al. 2013 (link)). The RNAs were purified using an RNeasy MinElute (Qiagen) kit and microinjected into one-cell-stage embryos at the indicated concentration. Microinjection was performed as described previously (Ishikawa et al. 2011 (link)).

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).

To construct TALEN, TAL repeats were assembled by the modified Golden Gate assembly method (Sakuma et al., 2013 (link)).
To construct sgRNA vectors for CRISPR-Cas9, two oligonucleotides corresponding to the 5’-side of exon 4 of the XBP1 gene (5'-taggCTGCGGACTCAGAAGACC-3' and 5'-aaacGGTCTTCTGAGTCCGCAG-3'), and two oligonucleotides corresponding to the 3’-side of exon 4 of the XBP1 gene (5'-taggTGCCTCCGCAGCAGGTGC-3' and 5'-aaacGCACCTGCTGCGGAGGCA-3') were annealed and ligated into BsaI-digested DR274 vector (Addgene). sgRNA vectors were linearized with DraI, purified by phenol chloroform extraction, and used as template to synthesize sgRNAs using T7 RNA polymerase.
TALEN and Cas9(D10A) expressing vectors were linearized with NotI, purified by phenol chloroform extraction, and used as template to synthesize capped mRNAs using the Message mMachine SP6Kit (Life Technologies, Gaithersburg, MD).
Synthesized RNAs were purified by RNeasy MinElute (Qiagen, Germany) and microinjected into one-cell stage embryos at the concentration of 50 ng/μl for both left and right TALEN, 100 ng/μl for Cas9(D10A), and 25 ng/μl for both sense and antisense strand sgRNA of Cas9. Injection was performed as described previously (Ishikawa et al., 2011 (link)).