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Generation of IVT templates

IK Igor Kogut
SM Sandra M. McCarthy
MP Maryna Pavlova
DA David P. Astling
XC Xiaomi Chen
AJ Ana Jakimenko
KJ Kenneth L. Jones
AG Andrew Getahun
JC John C. Cambier
AP Anna M. G. Pasmooij
MJ Marcel F. Jonkman
DR Dennis R. Roop
GB Ganna Bilousova
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Production of in vitro transcription (IVT) templates for mod-mRNA generation was adapted from Warren et al14. Briefly, plasmids used as template for PCR for IVT template preparation were obtained from Addgene: pcDNA3.3_KLF4 (catalog # 26815); pcDNA3.3_OCT4 (catalog # 26816); pcDNA3.3_SOX2 (catalog # 26817); pcDNA3.3_c-MYC (catalog # 26818); pcDNA3.3_LIN28A (catalog # 26819); pcDNA3.3_d2eGFP (catalog # 26821). Additional plasmids with inserts encoding mWasabi, human NANOG, and M3O (pcDNA3.3_mWasabi, pcDNA3.3_NANOG, pcDNA3.3_M3O) used for IVT template preparation were cloned using the following Addgene plasmids: pTEC15 (mWasabi, catalog # 30174); pGEM-NANOG (catalog # 16351); pMXs-hM3O-IP (catalog # 46645). IVT templates were PCR generated using listed plasmids as templates. Forward primer is 5′-TTGGACCCTCGTACAGAAGCTAATACG-3′ and reverse primer used to introduce 120 polyA tail sequence is 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTCCTACTCAGGCTTTATTCAAAGACCA-3′. These primers were synthesized by Integrated DNA Technologies. Reverse primer was synthesized as Ultramer oligos at a 4 nmol scale.

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