Cleancap reagent ag 3 ome

Template DNAs were prepared by PCR using PrimeSTAR Max DNA Polymerase (Takara Bio) and the primers listed in Table S1, and the Monarch PCR & DNA Cleanup Kit (New England BioLabs Japan Inc.) was used to purify the PCR products. Template DNA sequences used in this study are shown in Table S2. The MEGAscript T7 Transcription Kit (Thermo Fisher Scientific K.K.) was used to transcribe mRNAs from the template DNAs. Additionally, 6 mM GTP, 6 mM CTP, and 6 mM ATP were used from the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific) with 6 mM N1-methyl-pseudoUTP (TriLink Biotechnologies, San Diego, CA, USA) and 4.8 mM CleanCap Reagent AG (3′ OMe) (TriLink Biotechnologies). After transcription, template DNAs were digested using TURBO Dnase (Thermo Fisher Scientific), and transcribed mRNAs were purified using Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, MA, USA). Next, the mRNAs were dephosphorylated using Quick CIP (New England BioLabs Japan) and purified using the RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan). The mRNAs were quantified using NanoDrop One (ThermoFisher Scientific K.K.), and their sizes were confirmed using the Agilent RNA 6000 Nano Assay and the Agilent 2100 Bioanalyzer (Agilent Technologies Japan Ltd., Tokyo, Japan).