Antarctic phosphatase

Clean PCR products generated with plasmid templates, purchased from GenScript, were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of ARCA; 3′-O-Me-m⁷G(5′)ppp(5′)G (Trilink Biotechnologies, San Diego, CA, USA); Guanosine-5′-triphosphate (GTP); Adenosine triphosphate (ATP); Cytidine triphosphate (CTP); Uridine-5′-triphosphate (UTP) (in case of synthetic mRNA) (Life Technologies, Carlsbad, CA, USA); and N1-Methylpseudouridine-5′-triphosphate (Trilink Biotechnologies) in the case of synthetic modRNA [24 (link)]. The mRNA was purified either with the MEGA clear kit (Life Technologies) according to the manufacturer’s instructions or using Amicon Ultra-4 Centrifugal Filter Unit 4 mL,10 kDa (Millipore Sigma, Burlington, MA, USA) and treated with Antarctic Phosphatase (NEB). Next, the mRNA was re-purified with the MEGA clear kit. The mRNA was quantified using a Nano Drop spectrometer (Thermo Scientific, Waltham, MA, USA), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM Tris-HCl and 1 mM EDTA. The open reading frame for the Luc and nGFP modRNA is listed below. Endogenous Luc mRNA is a mixture of mRNA enriched with luciferase gene, obtained by isolation of RNA form 4T1-Luc cell line (ATCC #CRL2539LUC2).