Hiscribe t7 rna synthesis kit

RNA probes were synthesized with the Hi-Scribe T7 RNA Synthesis kit (NEB) using 0.5μg of PCR amplified template DNA (See S10 Table for primer sequences). 100pmol of RNA probes were then biotinylated using the Pierce RNA 3’ end biotinylation kit (Thermo Fisher) and purified according to manufacturer’s instructions. Biotinylated RNA probes were re-suspended in 100μl of high salt buffer (0.5M NaCl, 10mM Hepes pH7.9). Approximately 0.4mg of streptavidin magnetic beads (NEB) were washed in high salt buffer and incubated with biotinylated probes on ice for 1–2 hours with occasional mixing. The beads were then washed three times with wash buffer (0.1M KCl, 10mMHepes pH 7.9, 0.1% Triton-X100). Washed beads were incubated on ice with 100μg retinal extract and 6μg competitor RNA in binding buffer (0.1M KCl, 10mMHepes pH 7.9, 5μg/μl heparin, 0.1% Triton-X100 and 20U RNAse Inhibitor) for four hours with occasional mixing. Beads were then washed three times with wash buffer and the bound proteins were eluted in wash buffer containing 20ng RNAse A. The Musashi proteins in the eluates were detected by western blotting using an antibody that reacts with both MSI1 and MSI2 (S8 Table).

S1m-sgRNAs were designed by combining the sgRNA scaffold with the S1m aptamer sequence^{74 (link)}. S1m gBlocks were annealed with Phusion polymerase (New England Biolabs) under the following thermocycler conditions: 98 °C for 30 s followed by 30 cycles at 98 °C for 10 s, and 72 °C for 15 s with a final extension at 72 °C for 10 min. S1m cDNA was annealed with Phusion polymerase (New England Biolabs) under the following thermocycler conditions: 98 °C for 30 s followed by 30 cycles at 98 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s with a final extension at 72 °C for 10 min. In vitro transcription was performed with the MEGAShortscript T7 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For guide RNAs for fibroblast transfection, in vitro transcription was performed using HiScribe T7 RNA synthesis Kit (New England Biolabs) according to the manufacturer’s instructions.

The highly conserved sequences of the feline calicivirus ORF-1 gene were selected by aligning the full-length FCV genomic RNA from GenBank (https://www.ncbi.nlm.nih.gov/) to design specific RPA primers containing T7 promoter by Prime Primer 6.0 software and BLAST (https://blast.ncbi.nlm.nih.gov/). The pGM-T (Amp) plasmid containing 133 bp FCV ORF-1 gene sequence (from 2418-2550 nt) from FCV strain SMU-F4-2020 (Genbank no. MW194991) were constructed using pGM-T Ligation Kit (TIANGEN Biotech, China). The designed primers, probes, and crRNA oligonucleotides were synthesized by Sangon Biotech, China. The crRNA oligonucleotides containing T7 promoter were annealed into dsDNA using an annealing buffer for DNA oligos (Beyotime, China) and HiScribe T7 RNA synthesis kit (NEB, China). The synthetic crRNA was digested with DNase I (NEB, China) at 37°C for 15 min and purified with Monarch RNA Purification Kit (NEB, China) according to the instruction manual. The concentration of synthetic crRNA was determined using a Qubit 3.0 fluorometer (ThermoFisher Scientific, USA). All sequences used in this study were shown in Supplementary Table 1. The primer binding sites were shown in Figure 1.

The 1260 bp complete coding sequence of SARS-CoV-2 Wuhan-Hu-1 strain N gene (GenBank: MN908947) was amplified from 2019-nCoV_N_Positive Control plasmid (IDT) using primers (IDT) SARS2N_F and SARS2N_R (S1 Table) in standard end-point PCR with Q5 DNA polymerase (NEB). The PCR band representing the N gene coding sequence was subcloned into plasmid pMiniT2.0 (NEB) with the 5’ end of the coding sequence proximal to the vector-provided T7 polymerase promoter and confirmed by Sanger sequencing. HiScribe T7 RNA Synthesis Kit (NEB) was used to produce the SARS-CoV-2 N gene single-stranded positive-sense RNA via in-vitro transcription from the pMiniT2.0 construct. The IVT RNA was digested with DNase1 (NEB) and purified using RNAClean XP (Beckman Coulter). Purified IVT RNA was eluted in nuclease-free water, adjusted to a final concentration of 1 μg/μl in nuclease-free water, and stored at -80°C.

A synthetic gBlock containing a T7 promoter, the SARS‐CoV‐2 genomic leader, TRS, and the first 154 bases of E gene was ordered from IDT. This gBlock was used as template for in vitro transcription reactions with HiScribe T7 RNA Synthesis Kit (New England Biolabs, Cat. #E2040S) following manufacturer instructions. RNA was transcribed for 16 h at 37°C, then DNase treated and purified using illustra G‐25 spin columns (GE Healthcare, Buckinghamshire, United Kingdom, Cat. #27532501). Concentration of the resulting RNA was determined first by NanoDrop spectrophotometer of two high‐concentration dilutions (approximately 1 µg/µl and 100 ng/µl) measured in duplicate followed by a dilution in PBS to an approximate concentration of 2 × 10¹¹ copies/ml, and then by reverse transcription droplet digital PCR (RT–ddPCR) system (Bio‐Rad, Hercules, CA, USA) of two low‐concentration dilutions (approximately 100 and 10 copies/µl) measured in duplicate with the Mills‐sgE primer/probe set.

DENV2 3’UTR was in vitro-transcribed from a pcDNA 3.1-Apt-PR6452-3’UTR vector containing T7 RNA Polymerase promoter sequence, streptomycin/tobramycin RNA aptamer sequence, DENV2 PR6452 3’UTR sequence [11 (link)]. Briefly, pcDNA 3.1-Apt-PR-3’UTR was linearized with XbaI (NEB), transcribed with HiScribe T7 RNA synthesis kit (NEB), treated with DNAse I (Thermo Fisher Scientific) and purified with phenol-chloroform extraction. RNA integrity was assessed on denaturing 1% agarose gel.
C6/36 cells were transfected with 1 μg of the 3’UTR RNA using Lipofectamine 3000 (Thermo Fisher Scientific). Two hours post-transfection, cells were rinsed twice with 1X PBS, fixed for 10 min with 4% of paraformaldehyde and processed for RNA-FISH, as described above.