Maxiscript t7 in vitro transcription kit

For alkaline treatment 1 μg of mtDNA purified by sucrose gradient was treated either with 100 mM NaOH for the indicated times or RNase H2 (in-house preparation) and Mung Bean Nuclease (New England Biolabs). DNA samples were loaded directly on 1 X Tris-borate EDTA, 1% agarose gels or first denatured at 95°C for 3 min. For the detection of multiple deletions 3 μg of total DNA was digested with an mtDNA single cutter (BglII) and loaded on a 0.6% TBE–agarose gel. After electrophoresis, 250 mA for 4 h, gels were washed either in 10 × SSC (for denatured samples) or 400 mM NaOH for 15 min twice (for non-denatured samples), blot transferred to nylon membrane overnight and then UV cross-linked, total energy 1200 × 100 μJ/cm². Membranes were pre-hybridized with 10 ml of hybridization solution (2 × SSPE, 2% SDS, 6 × Denhardts reagent, 5% Dextran Sulfate) for 30 min at 55°C. Riboprobes were synthesized from PCR products incubated with ATP, CTP, GTP and α-³²P-UTP and T7 RNA polymerase at 20°C for 2 h (Ambion Maxiscript T7 In vitro Transcription Kit). After overnight hybridization at 55°C the membrane was washed repeatedly with 0.1 × SSPE, 0.5% SDS until there was no signal in the wash solution and exposed to X-ray film.

For Cas9 mRNA in vitro transcription, plasmid MLM3613 was obtained from Addgene (https://www.addgene.org/42251) and was linearized using PmeI. Linearized plasmid was in vitro transcribed using mMESSAGE mMACHINE T7 ULTRA kit (Ambion, Life Technologies) following the protocol of the manufacturer. After transcription, a poly(A) tail was added to the 3’ end of the capped mRNA using the Poly(A) Tailing Kit (Ambion, Life Technologies). The Cas9 mRNA was purified with MEGAclear Transcription Clean-Up Kit (Ambion, Life Technologies). As template for guide RNA expression from the T7 RNA promoter, plasmid DR274 was obtained from Addgene (https://www.addgene.org/42250). ECFP targeting guide sequences 13sgRNA and 35sgRNA were inserted into DR274 as described above. sgRNAs were in vitro transcribed from the DraI-digested sgRNA expression vectors using the MAXIscript T7 in vitro Transcription Kit (Ambion, Life Technologies). Resulting sgRNAs were purified with MEGAclear Transcription Clean-Up Kit (Ambion, Life Technologies).

Luciferase reporter mRNAs were generated using MAXIscript T7 in Vitro Transcription kit (Ambion) according to the manufacturer’s protocol in the presence of the cap analog. PCR products encoding a T7 promoter followed by luciferase and a poly(A) sequence were used as templates for in vitro transcription. 4T1 cells were seeded on a 24-well-plate and cultured overnight. Cells were transfected with capped HSV-ICP0-5’UTR-Rluc-pA, HSV-TK-5’UTR-Rluc-pA, IRF7-5’UTR-Rluc-pA or Rluc-pA together with capped Fluc-pA as a transfection control using Lipofectamine (Invitrogen). Cells were treated with 1μM PP242 for 24 hours and lysed 24 hours post transfection. Luciferase activities were determined using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instruction.

Guide RNAs from DraI-linearized pDR274 templates were synthesized using the MAXIscript T7 in Vitro Transcription kit (Invitrogen) following an established protocol [28 ]. The concentrations of sgRNAs were determined using a Nanodrop spectrophotometer, and the quality of the RNA was verified by agarose gel electrophoresis. 70 ng of each sgRNA directed against a specific gene, and 3 μg of dCas9 protein (Integrated DNA Technologies; diluted to 1 mg/mL) were incubated at 37C for 10 min to form a mixture of dCas9:sgRNA complexes. Control samples contained dCas9 protein without sgRNAs. All injection samples were adjusted to a total volume of 10 μL using nuclease-free water and contained 0.2% phenol red. Approximately 1 nL of the dCas9:sgRNA mixture or dCas9 control was injected into one-cell zebrafish embryos. 30–60 embryos were injected with a single type of mixture per injection day.