Megaclear spin columns

PTEN-mRNA and TRAIL-mRNA were synthesized in vitro, as previously described.⁴⁰ (link) Briefly, the human 5′UTR with Kozak sequence and 3′UTR sequence were commercially synthesized by Integrated DNA Technologies (Coralville, Iowa,USA) and sub-cloned into pcDNA3.3. The MEGAscript T7 Kit (Thermo FisherWaltham) was used to synthesize mRNAs, whereas m7GpppG was replaced with Anti-Reverse Cap Analog (ARCA) cap analog (New England Biolabs) and cytidine and uridine were replaced with 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink BioTechnologies), respectively. Reactions were sustained for 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5′-triphosphates. The synthesized mRNAs were purified with MEGAclear spin columns (Ambion) and quantitated with NanoDrop (Thermo Scientific).

We purchased pcDNA3.3-SOX2 and pcDNA3.3 enhanced green fluorescent protein (EGFP) from ADDGENE (Cambridge, MA, USA), and mRNA-SOX2 was synthesized as previously described^{34 (link)}. In brief, plasmid DNAs were used as the template for poly-(A) tail polymerase chain reaction (PCR). The forward and reverse primers 5′-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3′ and 5′-T (120)-CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3′ were used. After generation of poly-(A) tailed DNA fragments, tail PCR products were purified using a PureLink PCR purification kit (Invitrogen, Carlsbad, CA, USA). RNA was synthesized using a MEGAscript T7 kit (Ambion, Carlsbad, CA, USA) with purified tail PCR product. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA. To generate unmodified mRNA, we made a Cap/NTP mixture using ATP, CTP, UTP, and GTP components. Reactions were incubated for 3–6 h at 37°C, and DNase and Antarctic phosphatase (New England Biolabs) were also added. Synthesized mRNAs were purified using MEGAclear spin columns (Ambion) according to the manufacturer’s protocol and quantitated with a NanoDrop spectrophotometer.