Ribomax large scale rna production systems t7 kit

TALEN mRNAs and Cas9 mRNAs/gRNAs were transcribed in vitro according to previously published protocols (Liu et al., 2012 (link); Yu et al., 2013a (link)). Specifically, pCS2-TALEN-L, pCS2-TALEN-R and pSP6-2sNLS-spcas9 plasmids with correct insertions were linearized and recovered as corresponding templates. Transcriptions were carried out following the instructions of the Sp6 mMESSAGE mMACHINE Kit (Ambion, USA). For Cas9 in vitro transcription, the poly (A) signals were added to the 3′ end of the capped mRNAs by E. coli Poly(A) polymerase Kit (New England BioLabs, USA). For the in vitro transcription of customized gRNAs, the DNA templates were obtained from the pMD19-T gRNA scaffold vector by PCR (Yu et al., 2013a (link)). The transcription was carried out using the RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega, USA). Each pair of purified TALEN mRNAs and the corresponding donor plasmid were mixed to a final concentration of 500 ng/µl for the mRNA and 700 ng/µl for the donor DNA, respectively; purified Cas9 mRNA, gRNA and donor plasmid were mixed to a final concentration of 750 ng/µl for the mRNA, 10 ng/µl for the gRNA and 700 ng/µl for the donor DNA, respectively.

To reconstitute XBP1u splicing in vitro, a vector encoding XBP1u downstream of T7 promoter was used as template for the splicing substrate (plasmids were a kind gift from Dr Fabio Martinon, Lausanne, Switzerland). The splicing substrate XBP1u mRNA was prepared using a RiboMAX Large Scale RNA Production Systems—T7 kit (Promega) at 37°C for 3 h. In vitro transcripts were purified using an RNeasy kit (QIAGEN). Flag-RtcB WT and Flag-RtcB Y306F proteins were purified from HeLa cells stably expressing the two proteins, respectively. Cell pellets were lysed in a buffer containing 30 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1.5% CHAPS supplemented with a cocktail of protease and phosphatase. Flag-tagged RtcB proteins were affinity-purified from the lysates with anti-Flag M2 affinity gel (Sigma-Aldrich), followed by extensive washes. The human recombinant IRE1 cytoplasmic domain was purchased from Sino Biological. The reconstituted in vitro XBP1u splicing assay was carried out at 37°C for 2 h in kinase buffer (2 mM ATP, 2 mM GTP, 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 1 mM MnCl₂, 5 mM β-mercaptoethanol). 1 μg XBP1u RNA, 250 ng IRE1 protein, and IP-purified Flag-RtcB on beads were used for a 50 μl splicing reaction. The spliced products were column-purified using an RNeasy kit (QIAGEN) for RT-PCR analysis. The PCR products were resolved on 3% agarose gel.

Mutants of dTSSK^−/−, Mst77F^−/−, and Mst33A^−/− flies were generated by CRISPR/Cas9-mediated mutagenesis as previously described^{84 (link)}. Briefly, in vitro transcription of Cas9 mRNA was performed using an Sp6 mMESSAGE mMACHINE Kit (Thermo Fisher Scientific #AM1340). In vitro transcription of the designed gRNAs was performed using a RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega #PR-P1320). Purified Cas9-mRNA and gene-specific gRNAs were mixed at final concentrations of 1 μg/μL and 50 ng/μL, respectively, and injected into w¹¹¹⁸ embryos. The mutants were verified by PCR and DNA sequencing. Primers used for gRNA construction, PCR, and sequencing are listed in Supplementary Data 3.