Abi3730xl platform

The structure of the HvCMF3 gene was determined by analysis of its cDNA. Total RNA was extracted from leaf material of a 3-day-old barley seedling (cv. Barke) using the Trizol reagent (Thermo Scientific, Wilmington, DE, United States) following the manufacturer’s instructions. Concentration of the RNA is measured by help of a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States) and further diluted to 1 μg/μL for downstream application. The prepared RNA was first treated with RNase-free DNase I (Fermentas, St. Leon-Rot, Germany) to remove potential DNA contamination; then used for cDNA synthesis applying the SuperScript^TM III First-Strand Synthesis System Kit (Thermo Scientific, Wilmington, DE, United States) following the manufacturer’s instructions. Next, RT-PCR was performed using primers that cover the HvCMF3 coding regions (Supplementary Table 1) as previously described (Li et al., 2019 (link)). RT-PCR products were purified using the NucleoFast^® 96 PCR Kit (Macherey-Nagel, Düren, Germany) and Sanger sequenced on an ABI 3730 XL platform (Life Technologies GmbH, Darmstadt, Germany). The HvCMF3 exon-intron-structure was revealed by alignment of the coding sequence to the corresponding genomic region.

Subsequent library quality inspection, computer sequencing, and data analysis were performed by the Shanghai Aishe Gene Company (China). Sequencing was performed using an Illumina HiSeq X10 high-throughput sequencer, and the results were compared with the RECQL reference sequence for mutation detection using the Burrows-Wheeler Alignment tool. Mutation sites were annotated using the Genome Analysis Toolkit (Broad Institute, Cambridge, MA, United States). All pathogenic mutations detected by next-generation sequencing were verified by Sanger sequencing on an ABI3730XL platform (Life Technologies) to rule out false positives.

To study the stability of recoded sequences in vitro, we passaged ZIKV variants (Figure 1) ten times in VERO cells. Cells were seeded in 24-well plates 24 h before virus inoculation at multiplicity of infection (MOI) of 0.01. After 1 h at +37°C, the inoculum was discarded and cells were washed three times with +37°C DPBS. Fresh culture medium was added and cells were incubated for 7 days at +37°C, 5% CO₂. Afterward, the supernatant was collected and used to inoculate cells for the next passage. After 10 passages, the supernatant was collected for RNA extraction (see below) and reverse transcription (Invitrogen SuperScript IV Reverse Transcriptase). The CpG enriched segments were amplified (Invitrogen Platinum PCR SuperMix, High Fidelity) and sequenced (Applied Biosystems ABI-3730xL platform) using both amplification and inner nested primers listed in Supplementary Table 1. Sequences from the 10th passage were compared to sequences from the reference virus stocks obtained on C6/36 cells at passage 2 after transfection.
For the in vivo stability assay, RNA was extracted from brains of C57BL/6 mice inoculated at one day of age with different ZIKV variants (Figure 1) and sampled at 21 days after inoculation (see neonatal mouse study). The CpG enriched segments were amplified and sequenced using both amplification and inner nested primers listed in Supplementary Table 2.