Ultrapure h2o

Briefly, uncapped RNA was prepared using 1 μg of linearized DNA template in a MEGAScript reaction (Ambion, UK) according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20°C, pelleted by centrifugation at 14,000 RPM for 20 min at 4°C, washed once with 70% ethanol, centrifuged at 14,000 RPM for 5 min at 4°C, and then resuspended in UltraPure H₂O (Ambion, UK). Purified transcripts were then capped using the ScriptCap m7G capping system (CellScript, Madison, WI, USA) and ScriptCap 2′-0-methyltransferase kit (CellScript) simultaneously according to the manufacturer’s protocol. Capped transcripts were then purified by LiCl precipitation, as detailed above, resuspended in UltraPure H₂O, and stored at −80°C until formulation.

Self-amplifying RNA encoding the pre-fusion stabilized SARS-CoV-2 was produced using in vitro transcription. pDNA was transformed into E. coli (New England BioLabs, UK), cultured in 100 mL of Luria Broth (LB) with 100 μg mL⁻¹ carbenicillin (Sigma Aldrich, UK). Plasmid was purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and the concentration and purity was measured on a NanoDrop One (ThermoFisher, UK). pDNA was linearized using MluI for 3 h at 37 °C. Uncapped in vitro RNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 2 h at 37 °C, according to the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation at −20 °C, centrifuged at 14,000 RPM for 20 min at 4 °C to pellet, washed with 70% EtOH, centrifuged at 14,000 RPM for 5 min at 4 °C and resuspended in UltraPure H₂O (Ambion, UK). Purified transcripts were capped using the ScriptCap™ Cap 1 Capping System Kit (CellScript, WI, USA) for 2 h at 37 °C, according to the manufacturer’s protocol. Capped transcripts were purified by LiCl precipitation as described above, resuspended in RNA storage buffer (10 mM HEPES, 0.1 mM EDTA, and 100 mg mL⁻¹ trehalose) and stored at −80 °C until further use.

Most of the chemical compounds were obtained from Sigma-Aldrich, Inc (St. Louis, MO) including serotonin hydrochloride (≥98%), selected biogenic amines (dopamine hydrochloride (≥98%), (±)-octopamine hydrochloride (≥95%), tyramine hydrochloride (≥98%), and histamine (97%)), agonists (5-methoxytryptamine (97%), α-methyl-5-HT, 1,(3-chlorophenyl)piperazine hydrochloride (99%), tryptamine hydrochloride (99%), quipazine maleate salt (≥98%)), antagonists (methiothepin mesylate salt (≥98%), yohimbine hydrochloride (≥98%), cyproheptadine hydrochloride sesquihydrate (99%), mianserin hydrochloride (≥98%), ketanserin tartrate salt (97%), and selective serotonin reuptake inhibitor, fluoxetine hydrochloride (≥98%). Stock solutions of the compounds were prepared with dimethyl sulfoxide (DMSO) for the in vitro experiments and ultrapure H₂O (Invitrogen) for the in vivo experiments, and stored at −20 °C. 4-chloro-DL-chlorophenylalanine ethyl ester hydrochloride (98%) (PCPA) was purchased from Alfa Aesar (Ward Hill, MA) and prepared in 10% sucrose immediately before use.

Cells grown to stationary phase were transferred to acidic media (pH 3.5) and grown to logarithmic phase. Cells were synchronized in G1 phase over two hours with the addition of 2 μg/ml alpha-factor (Genscript) every hour. Cells were washed twice with sterile water. 2.5×10⁸ yeast cells were pelleted, resuspended in 1 ml 60% methanol, and disrupted by 5 consecutive freeze and thaw cycles using liquid nitrogen and warm water, followed by incubation at -20°C for 90 minutes and boiling at 100°C for 3 minutes. The lysate was centrifuged at 19000×g for 15 minutes and the supernatant frozen in liquid nitrogen. Methanol was evaporated in a SpeedVac (Thermo Scientific) and the residue was rehydrated in 100 μl Ultra-pure H₂O (Invitrogen, GIBCO). Determination of cellular dNTP concentration was performed as earlier described [86 (link)]. Each extraction was performed at least in triplicate.

Two distinct Locked nucleic acid (LNA) ASOs, RNV104L and RNV124, complementary to different regions of GHSROS (see Fig. S6), were designed in-house and synthesized commercially (Exiqon, Vedbæk, Denmark). The ASOs contained two consecutive LNA nucleotides at the 5′-end and three consecutive LNA nucleotides at the 3′-end –in line with gapmer design principles. The LNA ASO sequences were as follows: scrambled control sequence: 5′-GC TTCGACTCGTAATCACCTA-3′; RNV124 (underlined bases denote LNA nucleotides): 5′-ATAA ACCTGCTAGTGTCCTCC-3′; RNV104L: 5′-GTTAACTTTCTTCTTCCTTG-3′. Lyophilized oligonucleotides were resuspended in ultrapure H₂O (Invitrogen) and stored as a 100 µM stock solution at −20 °C. Briefly, LNA-ASOs were diluted to 20 µM in OptiMEM I Reduced Serum Medium (Invitrogen) and cultured cells were transfected according to the manufacturer’s instructions. Cultured cells were incubated at 37 °C in 5% CO₂ for 4 h, before 500 µl growth medium, containing 30% FCS, was added to the serum-free medium. The cells were transfected for 24–72 h and GHSROS levels assessed by qRT-PCR.