Depc water

All TRIzol-treated samples were stored at -70°C until use. Samples were thawed on ice and processed in accordance with manufacturer instructions using chloroform, 100% isopropyl alcohol, and 75% ethanol with the following modification: RNA was precipitated with 1 μl GlycoBlue (ThermoFisher). Precipitated RNA was re-suspended in 50 μl DEPC-treated water (Ambion). All samples were frozen and thawed on ice prior to running qRT-PCR.
Viral RNA was quantified by qRT-PCR in technical triplicates. A final reaction volume of 20 μl was used containing 10 μl serum or other sample RNA, 6.25 μl 4× TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems), 9 pmol reverse primer, 9 pmol forward primer, 10 μM probe, and 1.75 μl DEPC water (Ambion). Primers and probe (S4 Table) were adapted from Pastorino and colleagues [67 (link)] who reported a sensitivity of 27 RNA copies and 1.2×10⁻² infectious doses per reaction. Amplification was performed in a StepOnePlus instrument (Applied Biosystems) under the following cycling conditions: 1 cycle at 50°C for 20 minutes, 1 cycle at 95°C for 2 minutes, 45 cycles at 95°C for 5 seconds, and 60°C for 1 minute. CHIKV genome copies were determined by using a standard curve derived from a 10-fold dilution series of CHIKV RNA from passage 3 stock virus quantified by spectrophotometry.

RNA of European eel samples collected at 3 dpi was extracted and sequenced as described by Bracamonte et al. (2019). Japanese eel samples collected at 3 dpi and 2 control and 2 treatment samples of the European eel collected at 23 dpi were stored in RNAlater. The remaining six samples of the European eel collected at 23 dpi and all Japanese eel samples collected at 23 dpi were shock‐frozen in liquid nitrogen and stored at −80°C. Storage condition was included as a factor in the relevant model (see below). RNA was extracted with TRIzol (Life Technologies) following the manufacturer's recommendations for fatty tissue with slight modifications. A TissueLyzer II (Eppendorf) was used to homogenize tissue in 850 μl TRIzol. After centrifugation, another 150 μl TRIzol and 200 μl chloroform were added to the supernatant. RNA was precipitated with 500 μl isopropanol and washed with 1 ml 75% ethanol. It was resuspended in 50 μl DEPC‐water (Life Technologies) and incubated on a heat block at 50°C for 2 min. Concentrations were measured with a 2100 Bioanalyzer (Agilent Technologies). Samples were diluted to 40 ng/μl in DEPC‐water, precipitated with 3 M sodium acetate and 100% ethanol. Library preparation and paired‐end sequencing (100 bp) were performed at Macrogen on an Illumina HiSeq4000. The number of raw reads per sample ranged from 14.6 to 30.8 M (Dryad Repository).

Freshly isolated scoliotic (n = 18 from 9 donors) and non‐scoliotic (n = 12 from 6 donors) chondrocytes were lysed with TRIzol (Life technologies). RNA was extracted according to the manufacturer's instructions and re‐suspended in DEPC water (Life technologies). 1 µg of RNA was reverse transcribed with the Reverse‐transcriptase kit (Life technologies). RT‐qPCR was performed on a QuantStudio 7 (Life Technologies) using PowerUp Sybr Green master mix (Life Technologies) and primers listed in Table 2. The results were analysed by the comparative CT method using ß‐actin as the reference gene. When compared to untreated or alarmin‐treated controls, the fold change was calculated using the 2^−ddCT method and normalized to ß‐actin.

RNA was isolated using Trizol (Invitrogen - Thermofisher Waltham MA, USA) and then DNAse-treated using RNase-free DNase Kit (Qiagen, Germantown, MD, USA). Total RNA extracts were reverse-transcribed using the SuperScript III First-strand Synthesis SuperMix System kit (Invitrogen) to generate cDNA. In brief, 800 ng of total RNA were mixed with 10 μL of 2x RT Reaction Mix and 2 μL RT Enzyme Mix and made up to a final volume of 20 μL with DEPC water (Invitrogen). Quantitative Real-Time (qRT)-PCR amplification was performed using the KAPA SYBR FAST qPCR Master Mix (2x) ABI Prism on a StepOnePlus Real-Time PCR system (Applied Biosystems). All primers were designed using the web-based program Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) in combination with PrimerBlast for validation of target specificity (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The thermal cycler was programmed as follows: Enzyme activation at 95 °C for 3′ followed by 40 cycles of denaturation (95 °C for 3 s) and annealing (60 °C for 30s) and finally, dissociation curve analysis. Primer efficiency was determined using linear modelling for the amplification curves with the LinReg software version 2015.4 [27 (link)]. Relative quantification was calculated using the Pfaffl method [28 (link)]. Primers targeting FLI1, ASB2 and GAPDH are listed in Table S1.