Abi high capacity cdna reverse transcription kit

RNA was isolated from tissue using E.Z.N.A HP total RNA isolation kit (Omega Biotech, Norcross, GA, USA) and cDNA reverse transcribed using ABI High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). Human- and mouse-specific gene primer sequences were obtained from primerbank [32 (link)] and are listed in Table S1. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and relative expression calculated by 2^−ΔΔCT as previously described [33 (link)].

Total RNA was extracted from murine tissues or cells by TRIzol reagent (Invitrogen, USA), and cDNA was extracted by an ABI High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. Quantitative real-time PCR was performed using the ABI StepOnePlus Real-Time PCR system (Applied Biosystem, Foster City, CA, USA). GAPDH was used as an endogenous control. Data were analyzed using 2^{- ΔΔCT}.

The FLAG tagged YTHDF2 cDNA was cloned out of pLEX-FLAG-YTHDF2 [13 (link)] into the HindIII/EcoRI sites of the pK-Myc vector, replacing the Myc tag. The SV40 VP1 gene was PCR cloned from either WT or VPm virus infected BSC40 cell cDNA, then cloned into the NotI (between the N'-FLAG and insert gene) and Cla I site (3' of the stop codon) of pK, replacing YTHDF2. pK-FLAG-VP1, and the m⁶A mutant pK-FLAG-VPm, along with the negative control pK-Myc vector, were co-transfected with a GST expression plasmid (pK-GST) into 293T cells with polyethylenimine (PEI) in 12-well plates. Transfected cells were harvested 2 and 3 days post-infection for Western blot or RNA analysis by qRT-PCR. RNA was extracted with Trizol (Invitrogen), treated with RQ1 DNase (Promega), then reverse transcribed to cDNA with the ABI High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher 4368814). Half of the DNase-treated RNA was "reversed transcribed" in the absence of reverse transcriptase and used as the RT- control. qPCR was done with the VP1 primers 5'-taagatggccccaacaaaaa-3' and 5'-tccttttatgacgagctttgg-3' and GAPDH primers 5'-tgggtgtgaaccatgagaag-3' and 5'-gatggcatggactgtggtc-3'. VP1 primers were specifically designed to avoid any m⁶A mutations.

Total RNA was extracted from murine tissues or cells by TRIzol reagent (Invitrogen, USA), and cDNA was extracted by an ABI High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. Quantitative real-time PCR was performed using the ABI StepOnePlus Real-Time PCR system (Applied Biosystem, Foster City, CA, USA). GAPDH was used as an endogenous control. Data were analysed using 2^{− ΔΔCT}. Primers used in the assay was listed in the Supplementary Table 2.