EMCV vcDNA from E14TG2a
Enrichment and sequencing of vDNAThe cytoplasmic fractions were separated and nucleic acids were extracted as mentioned above. The customized panel containing probes that cover the full-length antisense EMCV genomic sequence was obtained from iGeneTech Bioscience (Beijing, China). The extracted nucleic acids were then hybridized with the probes using the TargetSeq enrichment kit (iGeneTech Bioscience, 324091-V3) according to the manufacturers instructions. Hybridized vDNA was captured by Hydrophilic Streptavidin Magnetic Beads (New England Biolabs, S1421) and then eluted with nuclease-free water on a magnetic separator immediately after heating at 98C for 5 min. The enriched vDNA was fragmented by a brief sonication. The ssDNA libraries were prepared using the Accel-NGS Methyl-Seq DNA Library kit (Swift Biosciences, 30024) following the manufacturers instructions. The libraries were sequenced on Illumina NovaSeq 6000 using the paired-end sequencing strategy. The DNA library preparation, high through-put sequencing and data analysis were conducted by Seqhealth Technology Co., LTD (Wuhan, China).