Ssrna ladder

Transcription reactions were prepared at room temperature in 20 μl transcription buffer (40 μM Tris-HCl pH 8, 5 mM DTT, 10 mM MgCl₂) with 2 mM NTP’s (NEB, N0450S) and 40 U/μl RNasin Plus (Promega, N2615). T7 (P266L) polymerase (20 (link)) was added to linearized plasmid DNA (25 ng/μl) at 37 °C, incubated for up to 2 h, and halted either by heating to >75 °C or addition of EDTA (12 mM final concentration). To detect RNA products by electrophoresis, samples were heated (90 °C, 5 min) in formamide loading dye (49% formamide, 5 mM EDTA, 0.05% xylene cyanol, 0.05% bromophenol blue) and loaded to a Novex TBE-Urea, 6% polyacrylamide gels (Invitrogen, EC68655BOX) to run in TBE (VWR, 97061-754) at 180 V for 50 min. A ssRNA ladder (NEB, N0364S) was used for molecular weight standards. Gels were stained with SYBR-Gold (Invitrogen, S11494) in TBE and imaged using a Bio-Rad ChemiDoc MP Imaging System. Densitometry was performed with Bio-Rad Image Lab Software v. 6.0.1. The ratio of RNA/DNA was calculated for each sample to control for loading differences. RNA and DNA were also quantified by Quant-iT Assay (Thermo Fisher Scientific, Q333140 and Q33120) according to manufacturer’s instructions.

Cells from CPCC 95 E. gracilis were removed from − 80 °C and resuspended in 2 mL of TRIzol™ Reagent (Invitrogen™). Samples were transferred to MP Biomedical Lysing Matrix C tubes and lysed using a MP Biomedical Fast Prep-24. The RNA was precipitated using 250  $\mu$ L of 0.8 M disodium citrate/1.2 M NaCl and 250 µL isopropanol. DNA was removed from the samples using a Dnase1 treatment before the RNA was precipitated again using the same RNA precipitation solution that was mentioned above and isopropanol. The quality of each sample was assessed by visualizing RNA following electrophoretic separation on a 1.5% BPTE agarose gel following glyoxal denaturation. Dnase-treated RNA and a ssRNA ladder were loaded on the gels (New England BioLabs, Whitby, Canada).

Messenger RNA purity and quantity was evaluated by NanoDrop spectrophotometry of a 1:10 dilution. For mRNA, an A₂₆₀/A₂₈₀ ratio of >2 and an A₂₆₀/A₂₃₀ ratio between 2.0–2.2 indicated acceptable purity for downstream application. Integrity of mRNA was analyzed on a 1% TBE agarose gel along with a ssRNA ladder (New England Biolabs, Ipswich, MA, United States), using RNase-free equipment and reagents. After samples were heat-treated at 65°C for 5 min, they were run with the gel tank on ice, at 90 V/11 cm electrode distance for approximately 30 min before visualization on a Vilber FUSION FX7 bioimager (Vilber Lourmat Sté, Collégien, France).

Plasmids carrying DI genomes were linearized using BsmBI or Esp3I. DNA was purified by phenol-chloroform extraction, back extracted with H₂O, passed through a 1 mL column of Sephadex™ G-50 Superfine beads (GE Healthcare, #17-0041-01), precipitated with 100% ethanol, washed with 70% ethanol, and resuspended in RNase-free water at a concentration of 1 µg/µL. Using linearized plasmid as a template, mRNA was synthesized in vitro using the T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320) according to the manufacturer’s instructions. RNA was then capped in a one-step reaction using Vaccinia Capping System (NEB, # M2080S) and mRNA Cap 2′-O-Methyltransferase (NEB, #M0366S). RNA cleanup was performed via phenol-chloroform extraction as described above for cleanup of linearized DNA. Concentrations were quantitatively measured by NanoDrop 1000 (Thermo Scientific). Cap 1 mRNA was analyzed alongside ssRNA ladder (NEB, # N0362S) on a 1% agarose-formaldehyde denaturing gel to confirm size and quality.

Phage genomes were isolated by the phenol/chloroform method described [20 (link)]. To determine the nucleic acid composition, 1 μg of each genome was incubated with DNase I or RNase A (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, separated on a 1% agarose gel and imaged. To determine ds or ssRNA in CAP3, RNase I_f (New England BioLabs, Ipswich, MA, USA) was added per manufacturer’s instructions to the ssRNA ladder (New England BioLabs), dsRNA ladder (New England BioLabs) and CAP3 genomic RNA. The reaction was stopped with 0.1% sodium dodecyl sulfate (final volume) and separated on a 2% agarose gel and imaged.

2 μg polyA+ RNAs were isolated using NucleoTrap mRNA Mini kit for polyA+ RNA extraction (Macherey-Nagel) and separated by 1% formaldehyde agarose gel. ssRNA Ladder (New England Biolabs) was used for marker. The agarose gel was prepared using NorthernMax Denaturing Gel Buffer (Ambion) and run using NorthernMax MOPS Gel Running Buffer (Ambion). RNA gel was washed two times with nucleotide-free water for 30 min each, followed by transfer in 10x SSC buffer to Amersham Hybond-N+ blot (GE Healthcare). RNA was then fixed by UV crosslinking with 120 mJ/cm². Labeling of random-primed probes was performed with the Prime-It II Random Primer Labeling Kit (Agilent) and a mammalian expression vector containing full-length FORCP cDNA. Hybridization was done overnight at 42°C in ULTRAhyb hybridization buffer (Ambion) as per the manufacture's instructions. Blots were washed at 42°C using 2X SSC+0.1% SDS and 0.1xSSC+0.1%SDS and imaged using a Phosphorimager.