Hiscribe t7 arca kit

To identify the potential trans-acting factors that might differentially bind to the canonical and variant 3′UTRs, an RNA pulldown and mass spectrometry assay was performed on in vitro transcribed canonical and variant forms of the IL18RAP 3′UTRs, V1 and V3. Briefly, The canonical, V1 and V3 biotinylated IL18RAP 3′UTR sequences (384 nucleotides) and the negative control (ultrapure water only) were produced by using an in vitro transcription HiScribe T7 ARCA kit (NEB, E2060S) following the manufacturer’s instructions. Briefly, 300 ng of purchased DNA template (50 ng μl⁻¹; Twist; Supplementary Table 15) was incubated with unlabeled ATP/GTP/CTP and 5% biotin-labelled UTP at 37 °C for 3 h. Next, DNase treatment was performed by incubating the reactions at 37 °C for 30 min and was followed by incubation at 65 °C for 10 min to terminate the reaction. The RNA products were purified by an RNA cleanup purification kit (Zymo Research, R1015). The concentrations of the purified RNA samples were measured by Nanodrop, and the expected length was analyzed by TapeStation.

pGEMHE plasmid constructs (Supplementary Data 1a) were linearized and 5′-capped mRNA was synthesized with T7 polymerase (NEB HiScribeT7 ARCA kit) according to manufacturer’s instructions. mRNA concentration was quantified using a Qubit 4 fluorometer (ThermoFisher) and RNA Broad Range assay kit (ThermoFisher; Q10211).

The plasmid pCMV-PE2 was cleaved with restriction endonuclease PmeI (100 µg DNA in 1 mL) for 4 hr at 37°C. The cleaved DNA was isolated by phenol-chloroform extraction and ethanol precipitation and resuspended at 500 µg/mL in TE buffer. The DNA was stored at –20°C. Eight 20 µL in vitro transcription reactions were set up using 1 µg of template DNA in each reaction using the New England Biolabs HiScribe T7 ARCA kit with tailing (E2060S; as per the manufacturer’s instructions) and incubated for 2 hr at 37°C in an incubator (not a temp block). Using eight 20 µL reactions, after transcription, DNase I treatment, and polyA tailing (as per the manufacturer’s instructions), the RNA was purified on four 50 μg New England Biolabs Monarch RNA cleanup columns (T2040L; as per the manufacturer’s instructions) and eluted in 25 μL per column RNase-free H₂O and pooled. The RNA was stored at –80°C and the yield from the total of eight reactions was ~200 μg purified PE2 mRNA by measuring A₂₆₀ on a Nanodrop 2000c spectrophotometer. Note that the nCas9-RT fusion mRNA is ~6500 nt. Detailed protocols can be found on protocols.io (https://doi.org/10.17504/protocols.io.b3fmqjk6).