Scriptmax thermo t7 transcription kit

Cas9 protein was prepared as described previously [18 (link)]. To prepare sgRNA for L-(I-SceI)-UC and sGFP-waxy-HPT reporter plants, the DNA fragment was amplified using the primers Guide-it scaffold Template and I-SceI-LUC Guide it or GFP-wx-Guide it, respectively. Each sgRNA was prepared using the ScriptMAX^Ⓡ Thermo T7 Transcription kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. To prepare the Cas9/sgRNA complex, Cas9 and sgRNA were mixed in a 5:1 weight ratio and incubated in 1× NEB3 buffer at 4°C for 30 min.

A total of 22 respiratory pathogens were used in our initial validation analysis of the specificity of the study primers. The strain names of these 22 pathogens are listed in Table 1. The Middle East respiratory syndrome coronavirus (MERS-CoV) EMC strain was kindly provided by Dr. Ron A. M. Fouchier, Erasmus Medical Center, Rotterdam, the Netherlands. The SARS-CoV Frankfurt 1 strain was kindly provided by J. Ziebuhr, University of Würzburg, Germany. The clinical isolates of human coronaviruses (HCoV)-HKU1, -OC43, -NL63, and -229E were described by previous studies.^{31 (link)–33 (link)}In vitro-transcribed RNA (GenBank accession number MN908947), synthesized using a ScriptMax Thermo T7 Transcription Kit (TOYOBO, Osaka, Japan), was used to determine the detection limit of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan).

Commercially available custom oligo pools (Twist Biosciences) were used to construct dsDNA templates of ribozyme libraries for in vitro transcription. The oligo pools were ordered with the T7 promoter and ribozyme sequence, which were amplified by PCR using primers Ligase-lib-f and Ligase-lib-r (Supplementary Data 1) and Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs (NEB)). The PCR product was column-purified using the DNA Clean & Concentrator-5 kit (Zymo Research). In vitro transcription was performed using a purified dsDNA template with a ScriptMAX Thermo T7 Transcription Kit (Toyobo) in a volume of 10 µL. After the transcription reaction, the solution was incubated for 10 min at 37 °C with a DNase-I (NEB) solution consisting of 2 µL DNase I (2 U/μL), 2 µL 10× DNase I Reaction Buffer, and 6 µL nuclease-free water. The RNA product was column-purified using the RNA Clean & Concentrator-5 kit (Zymo Research).

The biotin‐pre‐let‐7s were transcribed and labeled using the ScriptMAX™ Thermo T7 Transcription Kit (TSK‐101; TOYOBO) and Biotin‐16‐UTP (#11388908910; Roche, Indianapolis, IN, USA) according to the manufacturer’s guidelines. PCRs were performed with the Template DNA, and primers are listed in Table S2.

Templates for in vitro transcription were amplified by primers containing T7 RNA polymerase promoter sequences from the vector containing circADARB1 fragment flanked by group I self-splicing introns of the T4 phage Td gene. circRNA precursor were synthesized by a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) and the incubation system was modified as previously described^{5 (link)}. In vitro transcribed RNAs were extracted by phenol-chloroform (pH ∼ 4.5), precipitated with ethanol and resuspended in RNase-free water. For circularization^{79 (link)}, the in vitro transcribed RNAs were incubated with T4 RNA ligase I (NEB) at 16 °C overnight according to the manufacturer’s recommendations. Circularized RNAs (and remaining linear RNAs) were then separated on 5% Urea PAGE gel. circRNA bands were excised from the gel and eluted overnight in gel elution buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaOAc, 1 mM EDTA, 0.25% SDS). After elution, the gel fragments were discarded by centrifugation at 15,000 × g 4 °C for 2 min. circRNA was then purified using Phenol-chloroform (pH ∼ 4.5) and precipitated with ethanol. dsRNAs were produced by in vitro transcription using ScriptMAX Thermo T7 Transcription Kit (TOYOBO, TSK101). DNA templates were produced by PCR using primers containing the T7 promoter sequence. Details for these primers, dsRNAs are provided in Supplementary Table 1.

pTD1-Ucp1L, pTD1-Ucp1S, and pTD1-Ucp1C were linearized by NotI digestion and subjected to RNA synthesis using ScriptMAX Thermo T7 Transcription Kit (TOYOBO). The yielded RNAs were collected using a RNeasy Mini Kit (QIAGEN) and used as RNA input for an immunoprecipitation assay, as described below.
HEK293T cells transfected with pcDNA3.1-3×HA, pcDNA3.1-3×HA-IMP2, pcDNA3.1-3×HA-IMP2^K438R, or pcDNA3.1-3×HA-IMP2^K438Q by the JetPRIME reagent for 24 h were lysed in IP buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM MgCl₂, 0.05% NP-40, 1 mM PMSF, and PIC) containing 10 mM nicotinamide and 1 μM TSA by ten passes through a 27-G needle. After centrifugation, the cleared lysates were subjected to immunoprecipitation overnight at 4 °C with anti-HA antibody beads (clone 4B2, Wako Pure Chemical Industries). The beads were incubated with IP buffer containing 5 ng/μL RNase A (Nippon Gene) at room temperature for 15 min, washed five times with IP buffer, and then incubated in IP buffer containing synthesized RNAs (for Fig. 8e, 2500 ng Ucp1L RNA; for Supplementary Fig. 6d, 1250 ng Ucp1L RNA + 1250 ng Ucp1C RNA or 1250 ng Ucp1S RNA + 1250 ng Ucp1C RNA). IMP2-bound Ucp1 RNAs were isolated according to the RiboCluster Profiler RIP Assay Kit protocol (MBL) and subjected to real-time qPCR.